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OLDER Australians are being warned to not double up on flu shots amid fears the over-65s vaccine won't protect them against a deadly strain of the virus.
The calls from health authorities follow reports of older people rushing out to get a second flu shot, concerned that the federal government's free vaccine for over 65s doesn't include the Brisbane B strain.
Originating in Brisbane, the virus was named the deadliest in the world by the World Health Organisation last year and is included in this year's four-strain vaccination designed for those under 65, along with two A strains and another B strain.
Over-65s have been receiving a three-strain trivalent vaccination that doesn't cover Brisbane B.
During last year's brutal flu season, 90 per cent of the 1100 influenza-associated deaths recorded were in the elderly.
But health experts say there is no need for seniors to double up on jabs.
Australian Medical Association president Tony Bartone said the over-65s jab remains the best protection for older people.
"The advice of experts such as the peak national clinical advisory group - the Australian Technical Advisory Group on Immunisation - is quite clear and specific," Dr Bartone said, "and there is no data suggesting people over 65 need to take both vaccines."
Short supply
DR Bartone said as well being unnecessary, doubling up could threaten vaccine supply.
"There have been multiple reports of vaccines running out," he said. "We really need to be very judicious in our use of resources and make sure we have adequate supplies."
The unprecedented demand for the seasonal vaccines has almost exhausted current flu vaccines available through the National Immunisation Program following concerted state and national campaigns to lift flu immunisation rates.
Dr Bartone said the timing of the immunisation rollout was critical, with the peak flu season expected between July and September. Any shortfall in available vaccines could delay the process.
"Anything which interferes with this timely vaccination program is singularly unhelpful," he said. "We have an opportunity and often don't get a second bite of the cherry to immunise the community."
AUSTRALIAN Chief Medical Officer, Professor Brendan Murphy, described getting another jab that includes the Brisbane B virus as "pointless". He said it would not give any better protection.
"Unfortunately there seems to have been confusion in the minds of some people in Queensland due to the name of the virus," Dr Murphy said. "It has nothing to do with what is happening in Brisbane but was named in 2008 when the virus was first identified."
He said the decision to leave the Brisbane B virus out of the over-65s vaccine was "very deliberate".
"The super-powerful vaccine for the over-65s comes only in three strains and it doesn't really matter as most elderly people only get the A strain and it is unlikely the Brisbane strain - the least important of all the strains - will be a particular problem in the Oceania region."
Dr Murphy said if it was, it was more likely to be in younger people.
"Stronger defence"
QUEENSLAND Health's medical director of the immunisation program, Dr Alun Richards, echoed the AMA's reassurances, saying global experts recommended the trivalent vaccine for over-65s for a number of reasons.
He said older people do not respond as well to standard flu vaccine as the immune system response to flu vaccine decreases with age.
"The enhanced vaccine is designed specifically to increase the immune system's response to vaccine, especially against influenza A (H3N2) strain, which is more common and severe in people aged 65 and older," he said.
Dr Richards said the enhanced vaccine provides a stronger defence against two A strains and one B strain.
"Australian surveillance data shows those aged 65 and older are affected more by A strains that are circulating in the community, than they are by B strains," he said.
"Research also shows that older people tend to have a level of immunity to B strains because of exposure to these strains in previous seasons.
"Although the enhanced vaccine contains one less B strain virus, the benefits of better and broader protection against the strains included will outweigh any potential loss of protection against the missing alternative B strain virus."
Dr Richards said while Queensland Health does not recommend obtaining the two vaccines, there were no serious safety concerns for anyone who has already received both.
What you need to know
THE National Immunisation Program provides free flu vaccines for the elderly, as well as other high-risk groups including pregnant women, people with chronic diseases and Indigenous Australians.
There are two free vaccines for over-65s which work in different ways - FluZone High Dose, a high dose version; and Fluad, a "super-boosting" jab with an extra ingredient to boost its effectiveness. Both contain three strains.
Anyone aged 65 and over who hasn't received their flu vaccination is advised to book an appointment with their GP.
Dr Richards said over-65s are at greater risk of serious complications from influenza infection, and have the highest influenza-associated death rates each year.


text:
Successful space missions can rarely be attributed to a single thing. Rather, they are the result of a system of systems: integrated elements functioning effectively in their individual roles and together with related components, then those systems interacting with and supporting other systems to form a collaborative whole—from the spacecraft itself to the engineering and research teams that design and build it.
An example is found in spacecraft power systems. Unlike a gas-powered car or a battery-powered laptop, most spacecraft are powered by multiple energy sources—such as photovoltaic panels, fuel cells, and batteries—working in tandem to ensure the spacecraft functions throughout the course of a mission. As with any system, the appropriate combination of elements and the method of their management are key to high performance and efficiency.
One initiative at Glenn Research Center, the Hybrid Power Management (HPM) program, focused on joining new and mature technologies for optimal power systems applications in space and on Earth, with the goal not only to develop ultra-efficient space power systems, but to advance HPM to address global energy issues. The HPM program emerged from Glenn's long history of electric vehicle research dating back to the 1970s, including the NASA Hybrid Electric Transit Bus (HETB) project in the 1990s, which was the largest vehicle to use supercapacitor energy storage.
Unlike batteries that store and discharge energy through chemical reactions, supercapacitors store energy electrostatically. In this way, supercapacitors charge and release energy more quickly than a battery, and unlike a battery, can tolerate up to 1 million charge and discharge cycles without wearing out. Though there were certain disadvantages compared to batteries, such as low energy storage, supercapacitors presented an interesting candidate to couple with batteries or other power sources in space applications or in hybrid electric land vehicles.
To help test the effectiveness of supercapacitors for power systems, NASA formed a unique partnership— perhaps the only one in the Agency's history to involve a dragster.
The dragster was named "Bad Amplitude," and it was battery-powered. Capable of achieving a speed of 127 miles per hour in a quarter mile, the dragster presented an ideal testbed for the supercapacitors NASA was studying. Through the NASA Illinois Commercialization Center (NICC), which at the time provided technology commercialization services for the state's businesses, Glenn partnered with the dragster's developer, NetGain Technologies LLC of Lockport, Illinois, in 2003.
The partnership also focused on another advanced concept: the development of a retrofit system for converting rear-wheel drive vehicles into gas/electric hybrids, installing an electric assist motor and using supercapacitors instead of batteries.
While the supercapacitors ultimately did not suit the dragster or the retrofit system, at the conclusion of the NICC grant, NetGain had provided NASA with significant data on supercapacitor use and had proven the viability of a hybrid retrofit system (HRS) using batteries. The company continued developing the HRS, including methods for coordinating the operation of electric motors and internal combustion engines. This NASA-derived work has now led not only to commercial HRS technology, but also, according to NetGain, to the world's most popular line of motors for electric vehicles.
NetGain Technologies' patent-pending Engine/Motor Interface System (EMIS) employs an electric motor inserted into the drive train of a standard, gas-powered vehicle to supply electrical assist power to the internal combustion engine. Through monitoring multiple engine performance parameters, EMIS strategically establishes the appropriate amounts of assist power to significantly enhance the vehicle's fuel economy. The system operates in the background, requiring no driver intervention or changes to typical driving habits.

subset: commoncrawl


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11:20 (AP) Charter schools have cleared their most difficult remaining legislative hurdle in Mississippi.
House members voted 62-56 today with no debate to approve a House-Senate agreement on House Bill 369.
Charter schools — public schools that agree to meet certain standards in exchange for less regulation — would have to be nonprofit entities. So would management companies.
Yesterday, senators agreed to the exact bill the House passed in January. The Senate must still approve it and send it to Gov. Phil Bryant.
Boards in districts graded A, B and C would get vetoes over charter schools in their boundaries. Mississippi grades districts on an A-F scale. Students wouldn't be allowed to cross district lines to attend a charter school in another district. That bar could impede charter schools in small districts.
06:54 (AP) — Faced with repeated warnings that changes could jeopardize support, Mississippi senators have agreed to the House version of a bill to expand charter schools.
An agreement between the House and Senate was filed 20 minutes before an 8 p.m. CDT deadline yesterday to work out differences between the chambers. The bill (House Bill 369) must still be agreed to by a majority of both the House and the Senate and signed by Gov. Phil Bryant.
The House bill would allow a seven-member board to approve up to 15 new charter schools a year. Boards in districts graded A, B and C would get vetoes over charter schools in their boundaries. Mississippi grades school districts on an A-F scale. No student would be allowed to cross district lines to attend a charter school in another district. That bar on crossing district lines could impede the creation of charter schools in districts with fewer students.
Charter schools — public schools that agree to meet certain standards in exchange for freedom from regulations — would have to be nonprofit entities. So would their management companies.
The House earlier voted for the bill 64-56 in the early morning hours of Jan. 24 after 10 hours of debate and bill-reading. However, one supporter of the bill has since died and another is recovering from a serious illness. It was the narrow margin of that vote that led House Speaker Philip Gunn, R-Clinton, to resist many of the changes that Lt. Gov. Tate Reeves wanted. House negotiators would not bend on Reeves' desire to limit the veto power to only school boards in A- and B-graded districts.
"Though we hoped for a bill that would not send the message that 'C' was OK in Mississippi, we agreed to compromise to give 125,000 Mississippi children an opportunity for success," Reeves, a Republican, said in a statement.
By Monday, the only issue that was in play was whether students would be able to cross district lines to attend a charter school in another district. Reeves favors free crossing of lines. The House offered to allow students who attend D- or F-graded schools within D- or F-graded districts to cross lines. But Laura Hipp, a spokesman for Reeves, said House leaders declined to guarantee they could pass the bill with that change.
"We asked on that offer if they could give us a letter in writing to guarantee they could pass the compromise. They said they could not," Hipp said, explaining why Reeves decided to stick with the original bill.
"We don't have the appetite or the votes to do a complete crossing of district lines," said Rep. Brad Mayo, R-Oxford, one of the three House negotiators.
Even a limited bill would still be a big victory for charter school advocates. Last year, charter schools died in a House committee
"I applaud House and Senate leaders for coming to an agreement and sending me a workable public charter school bill," Bryant said in a statement. "I have long supported public charter schools and believe in the opportunities they give our children."
Current Mississippi law doesn't contemplate new charter schools, only conversions of existing schools that don't meet state standards for three consecutive years. A total of 35 of the state's 1,000-plus schools are eligible to be converted to charters this year, but only if a majority of parents petition and win approval from the state Board of Education.
Under the charter school proposal, a separate seven-member authorizing board would solicit and approve charter school applications, for both new schools and conversions. The board would begin operations Sept. 1 and seek proposals Dec. 1. Charter schools would be authorized for five-year terms.
… we'd like to ask for your support. More people are reading the Mississippi Business Journal than ever before, but advertising revenues for all conventional media are falling fast. Unlike many, we do not use a pay wall, because we want to continue providing Mississippi's most comprehensive business news each and every day. But that takes time, money and hard work. We do it because it is important to us … and equally important to you, if you value the flow of trustworthy news and information which have always kept America strong and free for more than 200 years.
If those who read our content will help fund it, we can continue to bring you the very best in news and information. Please consider joining us as a valued member, or if you prefer, make a one-time contribution.Click for more info


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Search engine giant Google is extending its Digital Garage 'bus tour' in Yorkshire to offer people of all ages digital training and advice through free workshops and face-to-face coaching.
The firm will be making 15 more stops across South Yorkshire in the weeks until Christmas. .
Delivered in partnership with Sheffield City Council, the double decker bus, which has been transformed into a digital training hub, kicked off its tour at the beginning of August in Ashbourne and has already visited over 30 locations in the region.
The free training on offer covers topics including 'Taking your first steps online', 'How to build your CV and personal brand online', 'Online safety and security' and 'Social media strategy', and ranges from beginner to intermediate levels - so anyone can take part. Visitors can book in advance or simply drop by for on-the-spot help.
The extension comes after overwhelmingly positive feedback from locals who have taken advantage of the free digital training in towns the bus has visited.
It follows the recent announcement that Google's Digital Garage on the high street, situated in the centre of Sheffield, is to stay open for an extra six months to April 2018.
Olivia Blake, Deputy Leader at Sheffield City Council said, "We're very pleased to hear the bus tour is going to be extended and how positively it's been received by residents across the region. The bus tour has opened up digital skills training to many more local people in South Yorkshire, and we urge everyone to make the most of it over the next two months."
An evaluation has found 88 per cent of attendees of Google Digital Garages have changed the way they run or promote their business online, with 68 per cent saying they had seen increased sales, bookings, web traffic or social media.
Anyone interested in the free coaching can see the full bus tour itinerary at g.co/DigitalGaragebus.


text:
(757) 627-5437
Web: view site
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To see our Privacy Policy please see the About Us section of this website.


text:
Devil's Brigade
Matt Freeman
Visit :
Celebrated Bay Area punk legend Matt Freeman of Rancid and Operation Ivy fame will release the long awaited, self-titled debut album for his side project Devils Brigade on August 31 via Hellcat Records. The bass player has assimilated a touring band to join him this fall when they hit the road with labelmates Street Dogs on a US tour.
Devils Brigade was conceived in 2000, when bassist Matt Freeman began writing songs with Tim Armstrong between Rancid tours. This led to a few EPs and some songs on Hellcat's Give Em The Boot compilation, but the project was put on the back burner when Rancid began touring and recording again. Years went by and after Rancid took a break from touring on the Let The Dominoes Fall record, Matt got the itch to reactivate the project with a new studio album.
"The original concept for this record was going to be a musical," says Freeman. "Tim had been kicking around an idea of doing a musical about the building of the Golden Gate Bridge. He had the title "Half Way To Hell Club." This was based on the name for the bridge workers who fell off the bridge while it was being built and fell into the safety net that was between them and the ocean. We grew up in the East Bay looking at that bridge and it has always been a part of our lives. Six of those songs ended up on the Devils Brigade record. The other songs were all original Devils Brigade songs from the first demo. We thought they were still good and we should record them again."
In early 2010, Matt and Tim entered the studio with DJ Bonebrake from the influential punk group X and recorded twelve quick tempered songs featuring Matt's signature, snarling vocals along with his snap, thumping upright bass, which shine through on roots driven punk tracks like "I'm Moving Through," "Who's Gonna Save Your Now" and "My Own Man Now."
"It was a great vibe in the studio," Matt adds. "The record has a real live and loose vibe. Loose in a good way. Tim's guitar style blended really well with DJ and everything clicked very quickly. We had a lot of fun making this record. I am going to take it out on the road whenever I can when Rancid is not doing anything."
Stay tuned to www.hellcat.com and www.myspace.com/devilsbrigadeofficial for updates and news regarding Devils Brigade.
Devil's Brigade
- 01 I'm Movin Through
- 02 My Own Man Now
- 03 Shakedown
- 04 Bridge Of Gold
- 05 Darlene
- 06 Ride Harley
- 07 Who's Gonna Save You Now
- 08 Desperate Times
- 09 Vampire Girl
- 10 Gentleman Of The Road
- 11 Protest Song
- 12 Half Way To Hell
- No: #80518
- US Rel. Date: 08/31/2010
- EU Rel. Date: 08/30/2010
- Type: Full


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Welcome to a very exciting church, a church where the hand of God is at work. It is not by accident that you are on our website. It is our goal and desire to introduce people to a personal relationship with Jesus Christ and to help them grow in their walk with Him. We wish to Evangelize the Sinner, Edify the Saved and Exalt the Savior. Wherever you are on your spiritual journey, it would be our privilege and honor to minister to you and your family.
All of our ministries are designed to accomplish and support this great commission. We strive to minister to people wherever they are regardless of the season of their life. As you review the following pages, you will find ministries aimed at children, youth, singles, couples, men, women and seniors. You will also find ministries relating to small groups, discipleship, music, worship and missions.
First Baptist Bentonville is a group of believers who are committed to the belief that the Bible is God's Holy Word, a perfect book, serving as our ultimate authority in all matters of life. We are actively involved in missions to Northwest Arkansas, across the nation and to the world. We are committed to the fact that Jesus Christ is the one way to eternal life in heaven.
We invite you to join us. It is our hope that you will prayerfully consider serving the Lord through First Baptist Church of Bentonville.
Bro. Phillip Smith
Senior Pastor


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sam edelman
NEW YORK — We keep hearing that this series doesn't matter all that much.
That when it comes to the Yankees and Rays, the final standings are just details.
Because when you get right down to it, the difference between finishing first
or second in the American League East this season is not terribly dramatic.
The winner heads to the postseason, and the loser probably will, too.
So, yeah, I can see where some people might be overlooking the moments in between.
But I bet you know better. I bet, if you've been paying attention, you understand what is at stake.
And even if you had forgotten, Rays owner Stuart Sternberg has not.
An hour before Tuesday night's game, Sternberg stood outside the third base
dugout at Yankee Stadium and talked about the idea that these were the
final few weeks of games for a team that will never be together again.
There was no discussion of departing players. No announcement of specific
payroll limits. Just an emphatic reiteration that the Tampa Bay Rays will not
be able to afford this same group of players in 2011.
"No question. Nothing can change it," Sternberg said. "Unfortunately there is
nothing that can happen between now and April that will change it,
unless Joe Maddon hits the lottery and wants to donate it."
The Yankees can act as if the AL East crown is some token piece of jewelry
because they've won it 17 times and will be in contention again next season.
For the Rays, it means something different. For Tampa Bay, it is an opportunity
that should not be ignored.
The Rays have a payroll in the $70 million range, and Sternberg insists that is not
sustainable. Tampa Bay is on pace to draw fewer than 1.9 million fans to
Tropicana Field this season, and teams with less than 2 million in attendance are typically not big spenders.
Recently leaked financial documents suggested the Rays turned a profit when
they had a miniscule payroll in 2007 and a smaller profit in 2008 when they
reached the World Series with a larger payroll. The Rays say they have lost
money since, and Sternberg says the infusion of a half-dozen postseason
gate receipts will not have a dramatic impact on the financial books this time.
"You could take the most optimistic scenario (and) we couldn't come close to
turning a profit this year," Sternberg said. "What happened in '08, we took
some revenues in, specifically having a seven-game series with the Red Sox,
and it did contribute to what we did in (payroll) in '09. But when people talk
about the lift that you get, it's really about how you do after the season in selling tickets for the next year.
"While we had gotten a pretty reasonable bounce in season tickets (in '09),
it just fell back, as I talked about this year in the beginning. (Sales) were terrible.
And I'm not optimistic that we're going to have the kind of lift that we had in '08,
which we needed to be double what it was. So I'm not optimistic that we're
going to have that kind of bounce. It could happen, but it's not going to."
In real-life terms, this means Carl Crawford's time in Tampa Bay could be done
in about a month. It means Rafael Soriano and Joaquin Benoit might be heading
out the bullpen door. It means Jason Bartlett could be traded, and perhaps Matt Garza, too.
It means a club the Rays have spent years creating and cultivating could be on
its way to being younger, cheaper and less impressive the moment the final game of 2010 is played.
Does that thought hurt, I asked Sternberg.
"Worse than it hurts anybody else," he said. "It's who we are; it's who we have to be.
We took every shot we could at not having to have this happen. Along with
the great fortune in 2008, along with what looks to be great fortune this year,
we put everything in place to have it happen, to put us in a position so we'd
be able to keep adding, keep signing, (doing) more long-term deals, stuff like that. It wasn't meant to be."
So don't be fooled into thinking these games don't matter.
Maybe from a historical standpoint they are only somewhat significant.
And maybe from the point of view of playoff seeding for a division
winner or a wild card they mean a little bit more. But for fans who
have followed this team from the early days of spring, these games
should mean the world. Almost as if a dear friend is preparing to move away forever.
- John Romano breaking my mf heart


text:
Hello, I"m trying to delete a text file but to no avail.
Here is the code
File myFile = new File("test.txt"); Scanner input = new Scanner(myFile); String oldText = ""; while(inputOld.hasNextLine()) { oldText = oldText + inputOld.nextLine(); } input.close(); myFile.delete();
File is still there when I look. Path is correct, and the weird thing is if I create a new File in the code and point it an existing file without using scanners I can delete it.
Example is:
File myFile = new File("test.txt"); myFile.delete()
and that works... So my assumption is that because the file is being used by program I cannot delete it. How can I force my program to stop using the file since .close() method appears to not be working...


text:
Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments.
Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.
Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
The fluorescence-based quantitative real-time PCR (qPCR)1 (1)(2)(3), with its capacity to detect and measure minute amounts of nucleic acids in a wide range of samples from numerous sources, is the enabling technology par excellence of molecular diagnostics, life sciences, agriculture, and medicine(4)(5). Its conceptual and practical simplicity, together with its combination of speed, sensitivity, and specificity in a homogeneous assay, have made it the touchstone for nucleic acid quantification. In addition to its use as a research tool, many diagnostic applications have been developed, including microbial quantification, gene dosage determination, identification of transgenes in genetically modified foods, risk assessment of cancer recurrence, and applications for forensic use(6)(7)(8)(9)(10)(11).
This popularity is reflected in the prodigious number of publications reporting qPCR data, which invariably use diverse reagents, protocols, analysis methods, and reporting formats. This remarkable lack of consensus on how best to perform qPCR experiments has the adverse consequence of perpetuating a string of serious shortcomings that encumber its status as an independent yardstick(12). Technical deficiencies that affect assay performance include the following: (a) inadequate sample storage, preparation, and nucleic acid quality, yielding highly variable results; (b) poor choice of reverse-transcription primers and primers and probes for the PCR, leading to inefficient and less-than-robust assay performance; and (c) inappropriate data and statistical analyses, generating results that can be highly misleading. Consequently, there is the real danger of the scientific literature being corrupted with a multitude of publications reporting inadequate and conflicting results(13). The publication(14) and retraction(15) of a Science "Breakthrough of the Year 2005" report provides a disquieting warning. The problem is exacerbated by the lack of information that characterizes most reports of studies that have used this technology, with many publications not providing sufficient experimental detail to permit the reader to critically evaluate the quality of the results presented or to repeat the experiments. Specifically, information about sample acquisition and handling, RNA quality and integrity, reverse-transcription details, PCR efficiencies, and analysis parameters are frequently omitted, whereas sample normalization is habitually carried out against single reference genes without adequate justification.
The aim of this document is to provide authors, reviewers, and editors specifications for the minimum information, set out in Table 1⇓ , that must be reported for a qPCR experiment to ensure its relevance, accuracy, correct interpretation, and repeatability. MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments, pronounced mykee) is modeled on similar guidelines drawn up for DNA microarray analysis(16), proteomics experiments(17), genome sequence specification(18), and those under discussion for RNA interference work(19)(20) and metabolomics(21), all of which are initiatives coordinated under the umbrella of MIBBI (Minimum Information for Biological and Biomedical Investigations, http://www.mibbi.org)(22). Compulsory inclusion of a common reporting language to allow data sharing is not proposed, although it is envisaged that a future update of these guidelines could include such a recommendation. Rather, these guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. They should be read in conjunction with recent publications that deal in depth with the issue of qPCR standardization(23)(24)(25)(26).
1. Nomenclature
A few terms require standardization to ensure clarification:
1.1 We propose that the abbreviation qPCR be used for quantitative real-time PCR and that RT-qPCR be used for reverse transcription–qPCR. Applying the abbreviation RT-PCR to qPCR causes confusion and is inconsistent with its use for conventional (legacy) reverse transcription–PCR.
1.2 Genes used for normalization should be referred to as reference genes, not as housekeeping genes.
1.3 TaqMan probes should be referred to as hydrolysis probes.
1.4 The term FRET probe (fluorescence resonance energy transfer probe) refers to a generic mechanism in which emission/quenching relies on the interaction between the electron-excitation states of 2 fluorescent dye molecules. LightCycler-type probes should be referred to as dual hybridization probes.
1.5 The Oxford English Dictionary lists only quantification, not quantitation; therefore, the former is the proper word.
1.6 The nomenclature describing the fractional PCR cycle used for quantification is inconsistent, with threshold cycle (Ct), crossing point (Cp), and take-off point (TOP) currently used in the literature. These terms all refer to the same value from the real-time instrument and were coined by competing manufacturers of real-time instruments for reasons of product differentiation, not scientific accuracy or clarity. We propose the use of quantification cycle (Cq), according to the RDML (Real-Time PCR Data Markup Language) data standard (http://www.rdml.org)(27).
2. Conceptual Considerations
To explain and justify the guidelines, we find it useful to review a number of key issues surrounding qPCR experiments:
2.1 Analytical sensitivity refers to the minimum number of copies in a sample that can be measured accurately with an assay, whereas clinical sensitivity is the percentage of individuals with a given disorder whom the assay identifies as positive for that condition. Typically, sensitivity is expressed as the limit of detection (LOD), which is the concentration that can be detected with reasonable certainty (95% probability is commonly used) with a given analytical procedure. The most sensitive LOD theoretically possible is 3 copies per PCR(28), assuming a Poisson distribution, a 95% chance of including at least 1 copy in the PCR, and single-copy detection. Experimental procedures typically include sample-processing steps (i.e., extraction) and, when required, reverse transcription. If the volume changes and the efficiencies of these steps are accounted for, the most sensitive LOD theoretically possible can be expressed in units relevant to the experiment, such as copies per nanogram of tissue. Experimental results less than the theoretically possible LOD should never be reported. It also follows that results of "0" are meaningless and misleading. LOD estimates in qPCR analyses are complicated by the logarithmic nature of Cq, because Cq is undefined when the template concentration is zero. Appropriate determination and modeling of the LOD in the qPCR is the focus of continued research(26).
2.2 Analytical specificity refers to the qPCR assay detecting the appropriate target sequence rather than other, nonspecific targets also present in a sample. Diagnostic specificity is the percentage of individuals without a given condition whom the assay identifies as negative for that condition.
2.3 Accuracy refers to the difference between experimentally measured and actual concentrations, presented as fold changes or copy number estimates.
2.4 Repeatability (short-term precision or intraassay variance) refers to the precision and robustness of the assay with the same samples repeatedly analyzed in the same assay. It may be expressed as the SD for the Cq variance. Alternatively, the SD or the CV for copy number or concentration variance may be used. CVs should not be used with Cqs, however(29).
2.5 Reproducibility (long-term precision or interassay variance) refers to the variation in results between runs or between different laboratories and is typically expressed as the SD or CV of copy numbers or concentrations. Cq values generated from different runs are subject to inherent interrun variation(30); hence, reporting interrun Cq variation is not appropriate.
Publications describing mRNA concentrations for target genes should make it clear precisely what the targets are. The transcripts of most human genes and many genes in other multicellular organisms are alternatively spliced(31)(32), and these splicing variants specify alternative protein isoforms, with variation in splicing patterns reported in different tissues or at different developmental stages. Consequently, single exon–based RT-qPCR assays may detect a number of splice variants, whereas intron-spanning primers may be more selective but may miss some splice variants altogether. Most recently, autosomal nonimprinted genes that display allelic imbalance in their expression have been described(33). Taken together, these findings imply that use of an RT-qPCR assay that simply targets one or at most 2 exons of an mRNA is no longer sufficient to describe the expression level of a particular gene. Consequently, sequence information for primers must be provided together with an assessment of their specificity with respect to known splice variants and single-nucleotide polymorphism positions documented in transcript and single-nucleotide polymorphism databases. For primer sets selected from the RTprimerDB database(34)(35), this is easily done by consulting the RTprimerDB Web site (http://www.rtprimerdb.org), which contains all the relevant information. For commercial assays, lot information and the providers' experimental validation criteria are required. The reporting of results for nonvalidated commercial assays and assays that have been validated only in silico are strongly discouraged.
It must be remembered that detection of the presence of an mRNA provides no information on whether that mRNA will be translated into a protein or, indeed, whether a functional protein is translated at all.
Immunohistochemistry, western blotting, or other protein-quantification methods are not always able to corroborate quantitative cellular mRNA data. It is now well established that there is frequently a lack of concordance between mRNA- and protein-concentration data(36), which is particularly true for mRNAs that specify proteins that are part of multifunction protein complexes(37). Finally, it has become clear that knowledge of the presence and function of specific microRNAs is as important to understanding gene expression as being able to quantify the mRNA species(38).
It is also necessary to be aware that most quantitative RNA data are not absolute, but relative. Thus, the reference genes or materials used for standardization are critical, and any assessment of the validity of an RT-qPCR experiment must also consider the appropriateness of the relative-quantification reference. Therefore, the development of universal reference DNA and RNA calibration materials, although very helpful(39)(40), will not be a universal panacea(41)(42).
Much of the variance in reported expression values produced in RT-qPCR experiments is not simply due to variation in experimental protocols but is caused by corrections applied by various data-processing algorithms, each of which makes its own assumptions about the data. Consequently, although qPCR has frequently been proclaimed a touchstone or a gold standard, in practice this "standard" is a variable one, and the reporting of results requires considerable sophistication of analysis and interpretation(43).
3. Research vs Diagnostic Applications
Applications of qPCR technology can be broadly divided into research and diagnostic applications. Research applications usually analyze a wide range of targets with a fairly low throughput and many different sample types. The main parameters that need to be addressed relate to assay analytical sensitivity and specificity, which in this context refer to how many target copies the assay can detect and whether the no-template controls (NTCs) are reliably negative, respectively.
In contrast, diagnostic applications usually analyze a limited number of targets, but require high-throughput protocols that are targeted at only a few sample types. Although all of the considerations that apply to research applications also apply to diagnostic assays, clinical-diagnostic assays have a number of additional requirements that need to be considered. These requirements include information on analytical sensitivity and specificity that in this context refers to how often the assay returns a positive result when a target is present and how often it is negative in the absence of the target. Furthermore, the accuracy and precision within and between laboratories is often monitored by external QC programs. Additional clinical laboratory requirements include criteria for generating reportable results, whether repeated measurements are made on samples, data on the resolution of false-positive/false-negative data, and the similarity of results from multiple laboratories that use the same and different technologies. Thus far, only a couple of interlaboratory comparisons have been performed, and both of these studies emphasized the need for standardization of qPCR diagnostic assays(44)(45). Another interlaboratory exercise is planned within the European Framework 7 project: SPIDIA (Standardisation and Improvement of Generic Pre-analytical Tools and Procedures for In-Vitro Diagnostics; http://www.spidia.eu).
4. Sample Acquisition, Handling, and Preparation
Sample acquisition constitutes the first potential source of experimental variability, especially for experiments targeting RNA, because mRNA profiles are easily perturbed by sample-collection and -processing methods. There is some suggestion that fresh tissue can be stored on ice without major effects on RNA quality and concentration(46), but although this supposition may be true for some mRNAs and tissues, it may not be universally applicable. Hence, it is better to be cautious. Consequently, it is important to report in detail where the tissue sample was obtained and whether it was processed immediately. If the sample was not processed immediately, it is necessary to report how it was preserved and how long and under what conditions it was stored.
A brief description of the sample is also essential. For example, microscopical examination of a tumor biopsy will reveal what percentage of the biopsy is made up of tumor cells, and this information should be reported.
Nucleic acid extraction is a second critical step. Extraction efficiency depends on adequate homogenization, the type of sample (e.g., in situ tissue vs log-phase cultured cells), target density, physiological status (e.g., healthy, cancerous, or necrotic), genetic complexity, and the amount of biomass processed. Therefore, it necessary that details of the nucleic acid–extraction method be provided and that the methods used for measuring nucleic acid concentration and assessing its quality be described. Such details are particularly crucial for RNA extracted from fresh frozen laser-microdissected biopsy samples, because variations in tissue-preparation procedures have a substantial effect on both RNA yield and quality(47).
5. QC of Nucleic Acids
5.1. rna samples
Quantification of RNA in the extracted samples is important, because it is advisable that approximately the same amounts of RNA be used when comparing different samples. There are several quantification procedures in common use, however, including spectrophotometry (NanoDrop; Thermo Scientific), microfluidic analysis (Agilent Technologies' Bioanalyzer, Bio-Rad Laboratories' Experion), capillary gel electrophoresis (Qiagen's QIAxcel), or fluorescent dye detection (Ambion/Applied Biosystems' RiboGreen). The methods produce different results, making it unwise to try to compare data obtained with the different methods(48). The preferred method for quantifying RNA uses fluorescent RNA-binding dyes (e.g., RiboGreen), which are best for detecting low target concentrations. In any case, it is advisable to measure all samples with a single method only and to report this information.
It is also important to test for and report the extent of genomic-DNA contamination and to record the threshold cutoff criteria for the amounts of such contamination that are tolerable. It is essential to report whether the RNA sample has been treated with DNase (including the type of DNase used and the reaction conditions) and to report the results from a comparison of Cqs obtained with positive and no–reverse transcription controls for each nucleic acid target.
It is also essential to document the quality assessment of RNA templates. The only situation in which this requirement does not apply is when the quantity of total RNA extracted is too low to permit quality assessment. This situation arises when RNA is extracted from single cells, plasma, other cell-free body fluids, some laser-captured samples, or clarified tissue culture medium. It also applies in cases in which extraction and RT-qPCR steps are performed as a continuous, single-tube experiment. Key information to report includes data on RNA quantity, integrity, and the absence of reverse transcription or PCR inhibitors. It is worth remembering that RNA degrades markedly in vivo, owing to the natural regulation of mRNAs in response to environmental stimuli(49). This source of RNA degradation is beyond the control of the researcher; one of its manifestations is that even high-quality RNA samples can show differential degradation of individual mRNAs.
The A260/A280 ratio must be measured in a buffer at neutral pH, but such measurement is not sufficient if the nucleic acid is to be used for quantitative analysis, especially when the aim is to measure minor differences (<10-fold) in cellular mRNA concentrations. The absorbance ratio does provide an indication of RNA purity, because the presence of DNA or residual phenol alters the ratio. Instead, one should provide gel electrophoresis evidence at the least or, better yet, results from a microfluidics-based rRNA analysis(50) or a reference gene/target gene 3′:5′ integrity assay(51). The advantage of the use of a Bioanalyzer/Experion system to calculate an RNA integrity number or an RNA quality indicator number is that these measures provide quantitative information about the general state of the RNA sample. It is important to bear in mind, however, that these numbers relate to rRNA quality and cannot be expected to be an absolute measure of quality. Use of a 3′:5′ assay requires that the PCR efficiencies of both assays be virtually identical(51) and not be subject to differential inhibition. This assay also necessitates the establishment of a threshold criterion that delineates the RNA quality sufficient to yield reliable results. Ideally, the assay should target a panel of "integrity reference genes," probably without introns, with a 3′:5′ threshold ratio of approximately 0.2–5. Clearly, further work is required to generate a universally applicable, cost-effective, and simple protocol for assessing RNA integrity.
Inhibition of reverse-transcription activity or PCR should be checked by dilution of the sample (preferably) or use of a universal inhibition assay such as SPUD(52)(53). If the RNA sample is shown to be partially degraded, it is essential that this information be reported, because the assay's sensitivity for detecting a low-level transcript may be reduced and relative differences in the degradation of transcripts may produce incorrect target ratios.
5.2. dna samples
In general, degradation is much less of an issue with DNA; however, it is important to be able to assess the extent of DNA degradation for forensic applications, i.e., in cases in which harsh environmental conditions at scenes of crimes or mass disasters or at sites involving missing-person cases may have degraded the chemical structure of DNA. The small amplicon size of qPCR assays helps to minimize assay-related problems, but methods have been developed that provide a quantitative measurement of DNA quality(54) and should be considered for such specialized purposes.
The potential for inhibition is a more generally applicable variable that must be addressed in a publication, and it is important to ensure that no inhibitors copurified with the DNA will distort results, e.g., pathogen detection and their quantification(55). Although such approaches such as spiking samples with positive controls(52) can be used to detect inhibition, different PCR reactions may not be equally susceptible to inhibition by substances copurified in nucleic acid extracts(56)(57). Consequently, it is better to routinely use dilutions of nucleic acids to demonstrate that observed decreases in Cqs or copy numbers are consistent with the anticipated result and to report these data.
6. Reverse Transcription
The reverse-transcription step introduces substantial variation into an RT-qPCR assay(58)(59). Hence, it is essential that a detailed description of the protocol and reagents used to convert RNA into cDNA be provided. This documentation must include the amount of RNA reverse-transcribed, priming strategy, enzyme type, volume, temperature, and duration of the reverse-transcription step. It is recommended that the reverse-transcription step be carried out in duplicate or triplicate and that the total RNA concentration be the same in every sample(58).
7. qPCR
The following information must be provided for qPCR assays: database accession numbers of each target and reference gene, the exon locations of each primer and any probe, the sequences and concentrations of each oligonucleotide, including the identities, positions, and linkages of any dyes and/or modified bases. Also required are the concentration and identity of the polymerase, the amount of template (DNA or cDNA) in each reaction, the Mg2+ concentration, the exact chemical compositions of the buffer (salts, pH, additives), and the reaction volume. The investigators must also identify the instrument they used and document all of the PCR cycling conditions. Because the consumables used affect thermal cycling, it is necessary to identify the use of single tubes, strips, or plates, and their manufacturers. The degree of transparency of the plasticware used, e.g., white or clear, is also important, because different plastics exhibit substantial differences in fluorescence reflection and sensitivity(60). When plates are used, the method of sealing (heat bonding vs adhesives) can affect the evaporation of samples at the plate perimeter and should therefore be documented.
Because PCR efficiency is highly dependent on the primers used, their sequences must be published. This requirement is perfectly feasible even with commercial primers, because there is a precedent for companies to make their primer and probe sequences available (http://www.primerdesign.co.uk/research_with_integrity.asp).
In addition, submission to public databases such as RTprimerDB is strongly encouraged; over time, these databases could become universal clearinghouses.
7.1. secondary structure
The structure of the nucleic acid target (e.g., stem and loop secondary RNA structure) has a substantial impact on the efficiency of reverse transcription and the PCR. Therefore, the positions of primers, probes, and PCR amplicons must take the folding of RNA templates into consideration. Sequences should be checked with nucleic acid–folding software, e.g., mfold for DNA (http://mfold.bioinfo.rpi.edu/cgi-bin/dna-form1.cgi) or RNA (http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-form1-2.3.cgi). Ideally, the folding structures should be made available to reviewers.
7.2. specificity
In silico tools such as BLAST or equivalent specificity searches are useful for assay design. Any appreciable homology to pseudogenes or other unexpected targets should be documented and provided as aligned sequences for review; however, specificity must be validated empirically with direct experimental evidence (electrophoresis gel, melting profile, DNA sequencing, amplicon size, and/or restriction enzyme digestion).
Algorithms for predicting an oligonucleotide's melting temperature (Tm) are useful for initial design, but the practical optimum temperature for annealing must be determined experimentally. Although primer optimization has become unfashionable, it is clear that poor annealing optimization has a large effect on assay quality(51). A marked presence of primer dimers produces a lower PCR efficiency in probe-based assays and may generate false positives in assays based on SYBR Green I. Some evidence for primer optimization should be provided to reviewers, ideally in the form of annealing temperature or Mg2+ gradients, and be presented as Cq values, plots of fluorescence vs cycle number, and/or melting curves(61).
7.3. controls and quantification calibrators
In addition to the no–reverse transcription control in RT-qPCR assays mentioned above, additional controls and/or quantification calibrators are required for all qPCR reactions. NTCs detect PCR contamination when probes are used and can also distinguish unintended amplification products (e.g., primer dimers) from the intended PCR products in SYBR Green I reactions. NTCs should be included on each plate or batch of samples, and conditions for data rejection be established. For example, NTCs with Cqs ≥40 could be ignored if the Cq for the lowest concentration unknown is 35.
Positive controls in the form of nucleic acids extracted from experimental samples are useful for monitoring assay variation over time and are essential when calibration curves are not performed in each run.
Quantification calibrators may be purified target molecules, such as synthetic RNA or DNA oligonucleotides spanning the complete PCR amplicon, plasmid DNA constructs, cDNA cloned into plasmids, RNA transcribed in vitro, reference RNA pools, RNA or DNA from specific biological samples, or internationally recognized biological standards (as they become available). Dilutions should be carried out into defined concentrations of carrier tRNA (yeast or Escherichia coli at 10–100 ng/μL). For detection of human pathogens, calibrators can be diluted into negative control sample RNA or DNA, or they can be diluted into healthy human plasma, after which lysis may be carried out in the presence of carrier tRNA. Serial dilutions of a particular template can be prepared as stock solutions that resist several freeze–thaw cycles. A fresh batch should be prepared when a Cq shift of 0.5–1.0 is detected. Alternatively, solutions for calibration curves can be stored for a week at 4 °C and then discarded.
For diagnostic assays, the qPCR should include an independently verified calibrator, if available, that lies within the linear interval of the assay. Positive and negative extraction controls are also recommended.
7.4. assay performance
The following assay performance characteristics must be determined: PCR efficiency, linear dynamic range, LOD, and precision.
7.4.1. PCR efficiency.
Robust and precise qPCR assays are usually correlated with high PCR efficiency. PCR efficiency is particularly important when reporting mRNA concentrations for target genes relative to those of reference genes. The ΔΔCq method is one of the most popular means of determining differences in concentrations between samples and is based on normalization with a single reference gene. The difference in Cq values (ΔCq) between the target gene and the reference gene is calculated, and the ΔCqs of the different samples are compared directly. The 2 genes must be amplified with comparable efficiencies for this comparison to be accurate. The most popular method is not necessarily the most appropriate, however, and alternative, more generalized quantitative models have been developed to correct for differences in amplification efficiency(62) and to allow the use of multiple reference genes(30).
PCR amplification efficiency must be established by means of calibration curves, because such calibration provides a simple, rapid, and reproducible indication of the mean PCR efficiency, the analytical sensitivity, and the robustness of the assay. Amplification efficiency should be determined from the slope of the log-linear portion of the calibration curve. Specifically, PCR efficiency = 10−1/slope − 1, when the logarithm of the initial template concentration (the independent variable) is plotted on the x axis and Cq (the dependent variable) is plotted on the y axis. The theoretical maximum of 1.00 (or 100%) indicates that the amount of product doubles with each cycle. Ideally, the CIs or SEs of the means of estimated PCR efficiencies are reported from replicated calibration curves.
Calibration curves for each quantified target must be included with the submitted manuscript so that this information can be made available to the reviewers; slopes and y intercepts derived from these calibration curves must be included with the publication. Differences in PCR efficiency will produce calibration curves with different slopes. As a consequence, differences between the Cq values of the targets and the references will not remain constant as template amounts are varied, and calculations of relative concentrations will be inaccurate, yielding misleading results.
Cq values >40 are suspect because of the implied low efficiency and generally should not be reported; however, the use of such arbitrary Cq cutoffs is not ideal, because they may be either too low (eliminating valid results) or too high (increasing false-positive results)(26).
7.4.2. Linear dynamic range.
The dynamic range over which a reaction is linear (the highest to the lowest quantifiable copy number established by means of a calibration curve) must be described. Depending on the template used for generating calibration curves, the dynamic range should cover at least 3 orders of magnitude and ideally should extend to 5 or 6 log10 concentrations. The calibration curve's linear interval must include the interval for the target nucleic acids being quantified. Because lower limits of quantification are usually poorly defined, the variation at the lowest concentration claimed to be within the linear interval should be determined. Correlation coefficients (r2 values) must be reported, and, ideally, CIs should be provided through the entire linear dynamic range.
The LOD is defined as the lowest concentration at which 95% of the positive samples are detected. In other words, within a group of replicates containing the target at concentrations at the LOD, no more than 5% failed reactions should occur. Low-copy PCRs are stochastically limited, and LODs of <3 copies per PCR are not possible. If multiple reactions are performed, however, accurate quantification of lower concentrations can be obtained via digital PCR(29)(63)(64). Indeed, concentration calibrators can be checked by limiting dilution to show that the percentage of failed and successful reactions follows a Poisson distribution.
7.4.4. Precision.
There are many explanations for variation in qPCR results, including temperature differences affecting the completion of annealing and/or denaturation, concentration differences introduced by pipetting errors, and stochastic variation. Precision in qPCR typically varies with concentration, decreasing with the copy number. Ideally, intraassay variation (repeatability) should be displayed in figures as SD error bars or as CIs on calibration curves with replicate samples. CVs should not be used with Cqs(29) but can be used to express the variance in copy numbers or concentrations. This technical variation should be distinguished from biological variation. Biological replicates can directly address the statistical significance of differences in qPCR results between groups or treatments. For diagnostic assays, it may also be necessary to report interassay precision (reproducibility) between sites and different operators.
7.5. multiplex qpcr
The ability to multiplex greatly expands the power of qPCR analysis(65)(66), particularly when applied to the simultaneous detection of point mutations or polymorphisms(67). Multiplexing requires the presentation of evidence demonstrating that accurate quantification of multiple targets in a single tube is not impaired, i.e., that assay efficiency and the LOD are the same as when the assays are run in uniplex fashion. This concern is of particular importance when targets of appreciably lower abundance are coamplified with highly abundant targets.
8. Data Analysis
Data analysis includes an examination of the raw data, an evaluation of their quality and reliability, and the generation of reportable results. Various data-collection and -processing strategies have been described, and a systematic evaluation has revealed that qPCR data-analysis methods differ substantially in their performance(68).
Detailed information on the methods of data analysis and confidence estimation is necessary, together with specification of the software used. The methods of identifying outliers and the disposition of such data must be specified. Documenting assay precision requires identification of the statistical methods used to evaluate variances (e.g., 95% CIs) and presentation of the corresponding concentrations or Cq values. Such information should include both repeatability and reproducibility data, if available. As discussed above, reporting of CVs for Cqs is inappropriate(29), because Cqs will always be lower (and therefore potentially misleading) than CVs calculated for copy numbers. Information must be provided on the methods used for assessing accuracy, including the statistical significance of reported differences between groups.
8.1. normalization
Normalization is an essential component of a reliable qPCR assay because this process controls for variations in extraction yield, reverse-transcription yield, and efficiency of amplification, thus enabling comparisons of mRNA concentrations across different samples. The use of reference genes as internal controls is the most common method for normalizing cellular mRNA data; however, although the use of reference genes is commonly accepted as the most appropriate normalization strategy(69), their utility must be experimentally validated for particular tissues or cell types and specific experimental designs. Unfortunately, although there is an increased awareness of the importance of systematic validation and although the potentially highly misleading effects of the use of inappropriate reference genes for normalization are widely known, these considerations also are still widely disregarded(70). Consequently, many molecular analyses still contain qPCR data that are poorly normalized.
Normalization involves reporting the ratios of the mRNA concentrations of the genes of interest to those of the reference genes. Reference gene mRNAs should be stably expressed, and their abundances should show strong correlation with the total amounts of mRNA present in the samples.
Normalization against a single reference gene is not acceptable unless the investigators present clear evidence for the reviewers that confirms its invariant expression under the experimental conditions described. The optimal number and choice of reference genes must be experimentally determined and the method reported(71)(72)(73).
8.2. variability
The inherent variability of biological systems may rival or exceed experimental differences between groups. This variation is often observed when many biological replicates are used to increase the statistical significance of the experiment. Although differences between biological replicates may be large, sufficient numbers may allow smaller experimental differences to be discerned. A recent publication provides a textbook example for handling such data and how to salvage biologically meaningful data from assays subject to high biological variation(74). Many factors contribute to experimental variation and influence the number of biological replicates necessary to achieve a given statistical power. Consequently, power analysis is useful for determining the number of samples necessary for valid conclusions.
8.3. qualitative analysis
The use of the PCR to detect merely the presence of a nucleic acid template, rather than to quantify it accurately, is referred to as qualitative PCR, which is widely used in pathogen diagnostics. The problem with qualitative/quantitative stratification of PCR methods is that an accurate yes/no answer requires information about the low-end sensitivity of the PCR assay. Consequently, even a qualitative assay should provide information about the assay's performance characteristics, especially with respect to the points discussed in sections 7.4.2. and 7.4.3.
The necessity for ensuring quality-assurance measures for qPCR and RT-qPCR assays is well recognized(25)(44)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86). The main difference between qPCR and conventional (legacy) PCR assays is the expectation of the former's potential to quantify target nucleic acids accurately. This difference must be clearly recognized, and one cannot assume that legacy PCR assays can translate directly into the qPCR format. Table 1⇑ provides a checklist for authors preparing a report of a qPCR study. Items deemed essential (E) are required to allow reviewers to evaluate the work and other investigators to reproduce it. Items considered desirable (D) are also important and should be included if possible, but they may not be available in all cases. Certainly, it is important to apply common sense: Compliance with all items on the checklist is not necessary for initial screening of expression signatures targeting hundreds of targets. Once a more limited set of targets (fewer than 20) has been identified, however, assay performance should be described as detailed by the checklist, which is hosted on http://www.rdml.org/miqe/.
In summary, the purpose of these guidelines is 3-fold:
1. To enable authors to design and report qPCR experiments that have greater inherent value.
2. To allow reviewers and editors to measure the technical quality of submitted manuscripts against an established yardstick.
3. To facilitate easier replication of experiments described in published studies that follow these guidelines.
As a consequence, investigations that use this widely applied technology will produce data that are more uniform, more comparable, and, ultimately, more reliable.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: J. Hellemans, Biogazelle; J. Vandesompele, Biogazelle; C.T. Wittwer, Idaho Technology.
Consultant or Advisory Role: R. Mueller, DermTech International.
Stock Ownership: R. Mueller, Sequenom; C.T. Wittwer, Idaho Technology.
Honoraria: None declared.
Research Funding: S.A. Bustin, Bowel and Cancer Research, registered charity number 1119105; J. Hellemans, Fund for Scientific Research Flanders; M. Kubista, grant agency of the Academy of Sciences, Czech Republic (grants IAA500520809 and AV0250520701); C.T. Wittwer, ARUP Institute for Clinical and Experimental Pathology and Idaho Technology.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
1 All essential information (E) must be submitted with the manuscript. Desirable information (D) should be submitted if available. If primers are from RTPrimerDB, information on qPCR target, oligonucleotides, protocols, and validation is available from that source.
2 FFPE, formalin-fixed, paraffin-embedded; RIN, RNA integrity number; RQI, RNA quality indicator; GSP, gene-specific priming; dNTP, deoxynucleoside triphosphate.
3 Assessing the absence of DNA with a no–reverse transcription assay is essential when first extracting RNA. Once the sample has been validated as DNA free, inclusion of a no–reverse transcription control is desirable but no longer essential.
4 Disclosure of the probe sequence is highly desirable and strongly encouraged; however, because not all vendors of commercial predesigned assays provide this information, it cannot be an essential requirement. Use of such assays is discouraged.
↵1 Nonstandard abbreviations: qPCR, quantitative real-time PCR; MIQE, Minimum Information for Publication of Quantitative Real-Time PCR Experiments; RT-qPCR, reverse transcription–qPCR; FRET, fluorescence resonance energy transfer; Cq, quantification cycle, previously known as the threshold cycle (Ct), crossing point (Cp), or take-off point (TOP); RDML, Real-Time PCR Data Markup Language; LOD, limit of detection; NTC, no-template control.
- © 2009 The American Association for Clinical Chemistry


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TCS, Cognizant widen lead over peers3 min read . Updated: 04 Feb 2013, 11:39 PM IST
TCS revenue up $1.4 billion in 2012 over 2011 while Cognizant sees revenue rising $1.2 billion, trumping Infosys, Wipro
TCS revenue up $1.4 billion in 2012 over 2011 while Cognizant sees revenue rising $1.2 billion, trumping Infosys, Wipro
Bangalore: A clear separation is emerging between leaders and laggards in India's information technology (IT) sector, placing Tata Consultancy Services Ltd (TCS) and Cognizant Technology Solutions Corp. at the top with incremental revenue of at least $1 billion in the last three years.
TCS's revenue in calendar years 2012 over 2011 increased $1.4 billion and Cognizant expects revenue to rise by $1.2 billion, trumping Infosys Ltd's incremental revenue of $406 million and Wipro Ltd's $382 million. Incremental revenue, a measure of market share growth, has evolved as the new benchmark for the Indian IT sector, with profit margins—the keenly tracked metric until now—eroding as firms push aggressively to gain a bigger share of their clients' outsourcing budgets.
The new benchmark also serves to highlight the changes in an IT industry that's trying to shed its dependence on traditional low-cost businesses to build their strengths in more specialized, and lucrative, tasks.
TCS's revenue over the last four years has nearly doubled, with its incremental revenue well above $1 billion consistently in the last three years, according to data compiled by Mint.
Cognizant, too, has made strides in terms of incremental revenue, consistently outpacing Infosys, Wipro and HCL Technologies Ltd, and staying above $1 billion in the past two years. The US-headquartered firm has forecast incremental revenue of $1.22 billion for 2012, just marginally below TCS's. Cognizant will report results on 7 February.
For Infosys, incremental revenue has dropped from over $1 billion in 2010 and 2011.
Importantly, traditional low-margin services such as software application development and maintenance are contributing less than half of the total business of IT firms such as TCS and Infosys, Mint's analysis shows.
Instead, TCS's percentage of revenue from the more lucrative consulting business has nearly doubled over the last four years, and was 3.2% in the October-December quarter, while revenue contribution from infrastructure services increased from 8.3% a year ago to 11.7%, indicating the bellwether of the Indian IT sector has been consistently grabbing market share from rivals.
TCS, Infosys and Wipro did not respond to email requests for comments for this story. Cognizant did not comment ahead of its results announcement.
TCS has also been increasing its share of new contracts, as evident in the steady decline in revenue from application development and maintenance (ADM) business that typically flows from existing customers. In the latest third quarter, ADM contribution to TCS's revenue dropped to about 42% from about more than half traditionally.
Infosys's ADM revenue share has declined 4% percentage points over the past four years, while revenue contribution from its consulting and system integration businesses has increased by about 10% percentage points.
HCL's ADM revenue contribution has been constant at around 50%, but contribution from its infrastructure services business has nearly doubled to 28.4%. For Wipro, though, revenue share from ADM increased from 43% four years ago to 53% in the latest third quarter, while infrastructure revenue share rose a modest 4% percentage points.
"Our channel checks suggest that for most customers where TCS is among the top vendors, it invariably is the lowest cost vendor. This helps it win more volume share with low risk to its existing billing rates," analyst Aniruddha Bhosale of Deutsche Bank Securities wrote in a note in October. "It can also exact operating efficiencies by having large exposure to customers."
Industry experts expect TCS to hold on to its crown in 2013. "I don't think there's a challenge to TCS still holding on to its No.1 position. The other two (Cognizant and HCL) will try to close the gap with TCS; and to do that, they'll have to win deals against TCS," said Arup Roy, principal research analyst at technology researcher Gartner Inc. "Let's not forget TCS is the biggest, base-wise, compared with the others; so they're quite heavy. So for them to post a much higher growth rate compared to the others is actually quite difficult."
At the end of 2008, during the height of the global recession, TCS had a 9.1% slice of the domestic IT services pie, the highest among the top five Indian service providers, data from IT industry lobby Nasscom show.
Four years on, TCS has increased its market share to 10.1%, as have Cognizant (from 3.7% to 6.4%) and HCL (from 2.8% to 4%). Analysts see Cognizant outpacing other Indian service providers, including TCS, this year in terms of revenue growth.
"Cognizant may continue growing faster, thanks to its niche in the healthcare sector. And the fact they are a US-based firm with a local sales force has also helped," said Hitesh Shah, director at IDFC Securities.
Cognizant's "strengths in traditional areas—app testing and development—are being augmented effectively with its growing consultative competency and focus on new tech areas such as mobility and analytics," said Phil Fersht, founder of HfS Research, an US-based outsourcing advisory firm.


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The Chestnut Hill Film Group, sponsored by the Chestnut Hill Local, presents John Guillerman's 1978 mystery, thriller "Death on the Nile" at 7:30 p.m., Tuesday, Feb. 15 at the Chestnut Hill Library, 8711 Germantown Ave. " Based on the novel by Agatha Christie, "death on the Nile" won an Academy Award for best costume design, and was nominated as best foreign film at the 1978 Golden Globes.
The film stars Peter Ustinov as detective Hercule Poirot, who is investigating the murders of an heiress (Lois Chiles) on the SS Karnak in Egypt. Other cast members include Bette Davis, Mia Farrow and Angela Lansbury.
The next film in the series is Hiroshi Teshigahara's 1964 classic, "Woman in the Dunes." Teshigahara was the first Japanese filmmaker to be nominated for the Best Director Oscar.


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by Nathan Lerner
Perhaps you are someone who would love to have a garden, but feel constrained by a lack of available space. If so, there is now an innovative methodology available that might resolve your conundrum.
As part of the Chestnut Hill Garden Festival on Sunday, May 1, Laurel Hill Gardens, 8125 Germantown Ave., will launch their approach to vertical gardens, which began as an experiment by French botanist, Patrick Blanc, who concluded in 1988 that plants could flourish on water and fertilizer, devoid of any soil.
Blanc developed a system that enabled vegetation to grow on walls, which were lined with felt. The craze swept through parts of Europe and Asia, but now vertical gardens are available in the U.S.
These come complete with either a sprinkler from below or a drip irrigation system installed above. A gutter system on the bottom serves as a reservoir and enables water to recirculate.
Phil Dominic, of Laurel Hill Gardens, pointed out some of the applications for vertical gardens: "Urban environments with small growing areas or no soil at all, apartment building balconies and kitchens with natural light exposure, as well as commercial building facades to reduce heating and air conditioning costs."
More information at 215-247-9490 orwww.laurelhillgardens.com/verticalgardens.
Nathan Lerner welcomes feedback at email@example.com.


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Introduction: Scott Phillips is a genealogical historian and owner of Onward To Our Past® genealogy services. In this guest blog post, Scott researches the history of an old acting professor of his—Jeff Corey—and discovers that filling in the blanks of Jeff's life story in turn fills in some blanks in his own life history.
If you follow my posts here on the blog for GenealogyBank.com, you read toward the end of my latest article "Finding the Historical Articles That Tell My Ancestor's Story" that I had discovered a one-line death notice for Jeff Corey. He was one of my favorite professors when I was a student in the "World Campus Afloat" program. While I remember him as my instructor, you might best recall him as Sherriff Ray Bledsoe in the movie Butch Cassidy and the Sundance Kid.
Finding Jeff's death notice led me to think back on many of the stories that this friendly, approachable, and talented professor shared with me when I was a student, and reminded me how important he once had been in my life. Sadly, I realized that although he once mentored me, he actually was a blank in my life history—I really didn't know very much about Jeff Corey.
These memories prompted me to undertake another search in GenealogyBank.com and see what else I might discover about Jeff. As usual, I wasn't disappointed and I was able to more fully document and add this person from my own life to my family's extensive family history and genealogy—filling in the blanks about Jeff's story in turn filled in a blank in my own life history.
The first thing I did, as any good genealogist does, is look for multiple copies of an individual's obituary. I was very happy to discover that, while my earlier find had been only that one-sentence death notice, more than a dozen other newspapers provided more extensive obituaries for Jeff Corey. As you might expect for an American actor, one of the best I found was in a Los Angeles newspaper.
Not only did this extensive obituary list some of Jeff's best known roles in movies such as Butch Cassidy and the Sundance Kid, In Cold Blood, and Little Big Man, it also listed some of his television credits in successful sitcoms such as One Day at a Time and Night Court. Then his life story got really interesting.
I found more information on a time in Jeff's life that he had only briefly touched on when we talked those many years ago as student and instructor. This was the period when Jeff Corey was blacklisted in Hollywood for more than ten years! His mistreatment was a result of Jeff's appearance before the infamous Senator Joseph McCarthy and the House Committee on Un-American Activities in the 1950s. Once again, GenealogyBank.com and its database of historical documents proved invaluable.
In GenealogyBank's U.S. Congressional Serial Set collection, I found the Annual Report for the Committee on Un-American Activities for the year 1952. It included Jeff Corey with the notation: "(Appeared Sept. 21, 1951, and refused to affirm or deny Communist Party membership.)" On the same page you can see many others who also refused to comply with the committee's demands.
This action was enough to get Jeff Corey blacklisted and banned from any work in Hollywood for more than ten years. I found Jeff's comment, related in his obituary, to be most interesting. He said "The only issue was, did you want to just give them their token names so you could continue your career, or not?" He chose not to name any others in Hollywood.
I think you could say that Jeff got the last laugh, though. While I am sure he missed out on a multitude of roles in those ten years—and he did tell me they were some very lean years—he became one of the most sought-after acting coaches in all of Hollywood!
This 1975 California newspaper article reported that some of Jeff's more notable students were such Hollywood superstars as Jack Nicholson, Anthony Quinn, Jane Fonda, and Kirk Douglas. Needless to say, I was truly impressed by this talented group.
Jeff's return to the "big screen" was noted in this 1961 Louisiana newspaper article.
Jeff continued to coach actors even after he returned to his career in acting. I found a wonderful quote praising Jeff by one of my favorite actors, James Coburn, published in this 1979 Ohio newspaper article.
After I finished my genealogy research on Jeff, I was pleased with how much information I had found and how much more I knew of this cherished professor. I was also happy because I had filled in a delightful segment in my own family history story—one I hope my children and grandchildren will someday enjoy reading as much as I did researching and writing it.
My closing advice is this: Don't overlook your own life stories while you are working on your genealogy. They can be great fun and lead to many surprising discoveries!


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I consult the dictionary of human behavior every day.
I had to load it into my brain and make it learn
that you open doors with hello and
that you close them with goodbye. I had to learn
the mechanics of when to smile, when to laugh.
(From my poem, "You Don't See It")
As a woman, I have been aware (painfully at times) of the expectations that Western society and culture has placed upon us, both past and present. I mentioned some of these expectations in my last post when I talked about Aspie women and our unique challenges navigating the social matrix. Some of those expectations are also applied, along with a few others, to women in the realm of romantic relationships. This week,I will discuss those expectations and the challenges that Aspie women might have meeting them when involved in a close relationship.


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JAK88 needed a new logo design and created a contest on 99designs.
2 winners were selected from 72 designs submitted by 19 freelance designers.
Clean, eye-catching logo needed for global tech network promoting openness, innovation, cutting edge technology and visionary ideas.
Open ePolicy Group (or OeG)
Desired Color Scheme:
Open to your ideas
Desired Style:
Clean, light and techy ... but with dynamic and entrepreneurial feel.
Accepted File Formats:
High quality image. 300 pixels per inch, preferably PSD with layers or vector Illustrator file. With fonts. Plus JPG & GIF
About Us: The Open ePolicy Group or OeG is a global technology group comprised of leading governments, companies and tech minds. Our focus is on how open technologies drive innovation and business transformation. Our first publication --- downloadable at http://cyber.law.harvard.edu/epolicy/roadmap.pdf --- has nice cover image that conveys integration of technology and nature, which we liked. In other words, more Apple than IBM in terms of design.
Our Audience: High-level government officials who use our work and tech companies that sponsor us. Our brand, members and work are global.
General Specs: The name Open ePolicy Group or OeG should be incorporated with a graphical logo. However, logo itself should be an image that is eye-catching, simple and brandable standing alone.
Both logo and font should be clean, cool and appeal to a global audience. It should convey feeling that we are about openness, innovation, cutting edge technology and visionary ideas.
Design Style: clean, light and techy ... but with dynamic and entrepreneurial feel to it.
Keywords: open, global, community, revolutionary zen.
Deliverable File Types: High quality image. 300 pixels per inch, preferably in Photoshop PSD hi-res format with layers or a vector Illustrator file. With fonts supplied. Plus a copy in JPG and GIF.
Usage: Logo is expected to serve as a key design element for business cards, website, presentations, blog and publications. Later professional design of business cards and a website is anticipated.
Designer warrants that submission is original. Please no stock photos, stock artwork, clip art, or elements from another website, company or logo. Upon full payment, I MUST own full copyright and will insist upon an indemnification from winning designer in the event any aspect of logo is claimed by another.
Individual feedback will be provided as promptly as possible.
Its a clean slate so let those creative synapses fire! This logo will be seen by influential officials, executives and leading technologists worldwide. Great exposure for us and you.
Find out how a design contest works or learn more about our logo design services.


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Walmart's octuparra droid exclusive - found today. 4 of them in the regular section mixed in with the $24.99 vehicles.I highly recommend - this thing is huge and really worth the $35. Nice and sturdy too.
Found the Y-Wing and the two new "Rookies" exclusive two packs at one of the local Wal-Marts today. While I had initially planned on ditching the Rex, the episode-accurate Rishi eel goo on his hand and wrist changed my mind.As for the Y-Wing: WOW.
I picked up the Octupara Droid.
Page created in 0.029 seconds with 19 queries.


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CAMBRIDGE, Mass. – MIT put on an offensive show this afternoon, banging out 18 hits and scoring runs in every inning but two as the Engineers rolled to a 19-3 win over visiting Worcester State University this afternoon. Every starter had at least one hit for MIT and sophomore Robert Bertucci (Bellville, Ill.) was strong in his first start of the season as the Engineers won their season-high fifth straight game.
It did not take long for MIT (16-13) to get on the board, pushing a run across in the bottom of the first inning. With one away sophomore Sean Karson (Winter Park, Fla.) drew a walk and then moved to second on a wild pitch before a single by sophomore Creed Mangrum (Sandy, Utah) moved him up to third. Junior Ricardo Perez (Vienna, Va.) then brought him in to score, lofting a sacrifice fly to center.
Perez drove in the next MIT run as well, doubling to right center in the third to bring around Mangrum, who had doubled ahead of him. Perez's hit brought in freshman Jared Kuzio (Franklin, Mass.) out of the bullpen for Worcester State (9-21) in relief of freshman starter, D.J. Colbert (Somersworth, N.H.). He struck out his first batter before walking freshman Hayden Cornwell (Newport Beach, Calif.) and then throwing a wild pitch that put runners on second and third. Junior James McKinney (Wakefield, R.I.) brought both runners home with a single to center to make it 4-0.
MIT's offense kept cranking out the runs, adding two more on an RBI single from Mangrum in the fourth and a pair of unearned runs in the fifth that came around to score on a throwing error on the Lancers shortstop. That was more than enough support for Bertucci, who did not allow a hit until the fourth. He picked up the win, going five innings and allowing just the one hit, striking out six and walking two.
MIT broke the game open in the sixth when the Engineers sent 13 batters to the plate, scoring eight times. Karson and sophomore Jon Rae (Palo Alto, Calif.) both drove in a pair of runs with singles, with freshman Parker Tew (Chapel Hill, N.C.) adding a pinch-hit, two-run double.
Worcester State broke into the scoring column in the seventh off the MIT bullpen. Junior Kurt Sabacinski (Worcester, Mass.) drew a leadoff walk before Joseph Carlier (Rockland, Mass.) was hit by a pitch. Freshman David Ruggiero (Peabody, Mass.) singled to center to plate the first run and freshman Jeff Bolio (Oxford, Mass.) later added a sac fly for the Lancers. Worcester State added another run in the eighth when Sabacinski was hit by a pitch before moving to second on a wild pitch. He then came around to score on a pinch-hit single by freshman Brian O'Connor (West Boylston, Mass.).
Senior Zach Carr (Chatham, N.J.) and freshman Phil Reiser (Alpharetta, Ga.) added RBI singles in the seventh for MIT.
MIT will open a key three-game NEWMAC series on Friday, taking on WPI in Worcester in a single game starting at 3:30 p.m. Worcester State will play again tomorrow when it travels to Becker College for a 4:00 p.m. game.


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|Stoneforge Tavern & Publick House, built in 1998 by William and Patricia Roland in Raynham Massachusetts, is named in part due to Raynham being the location of the first working iron forge in America. The Raynham location was built with a single purpose in mind; a guest can come to the same location three different times and get three different dining experiences. Learn More About Stoneforge Restaurants
The Stoneforge Tavern in Raynham, Massachusetts. Casual dining with creative cuisine and cocktails.
The Publick House in Raynham, Massachusetts. "Smart casual" dining featuring signature beef, seafood, and saute dishes.
The Stoneforge Grill located in
So. Easton, Massachusetts offers classic New England cuisine with sophisticated global influences.
Stoneforge Ballroom is
conveniently located in Raynham, Massachusetts, off of route 44. Easily accessible to routes 24 and 495. Adjacent to a major hotel.


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Drought claims will drive up corn and soybean insurance payouts this year, but one insurance executive isn't discouraged.
"Crop insurance is a great business," Evan Greenberg told equity analysts in a July 25 conference call after the company reported first-half earnings. "Over any period of time it has been very good," said Greenberg, chairman and chief executive officer of ACE Limited, a multiline property and casualty insurer that provides commercial and personal insurance through operations in 53 countries. Rain and Hail Agricultural Insurance is one of the ACE companies.
Greenberg said crop insurance earnings are under pressure now, "But they're still projected to be reasonable."
The company's operating income in the first half of 2012 rose from a year earlier, but ACE trimmed its income outlook for the second half because of the higher expected payments to crop insurance policy holders.
Crop insurance profits over several years and geographic diversification help keep this year in perspective.
"The health of the crop insurance industry as a piece of the broader insurance industry, as best we know, is quite good," said Dave Miller, director of research and commodity services at Iowa Farm Bureau. Corn and soybean crop insurance payouts will be high for the 2012 crops, but insurance companies spread their risks across a wide geographic area and broad insurance markets—with backing from the federal government.
Greenberg said his company's provision for crop insurance payouts in the third quarter assumes the highest probable outcome, based on current weather and crop conditions. "We don't expect it is worse than that," he said. "But what we do know is there's still that question: Could it be worse? No one can predict the weather."
Greenberg declined to estimate the insurance industry's potential payments on crop insurance this year. "It's way too early to know," he said. "We have no idea."
Different States at Risk
This year producers and insurers are dealing mainly with major crop damage from drought in the Midwest. Last year, they faced floods, drought, tropical storm damage, and tornado losses in other regions.
Last year, the ratio of claims paid to premiums paid reached 0.9 on a national scale, reports USDA's Risk Management Agency, which manages crop insurance operations for the Federal Crop Insurance Corp. Federal law requires an expected national loss ratio of no more than 1.0, meaning that the amount of premiums at least equals claims paid.
Some states hit hard by drought this year reported low claims relative to premiums last year. The 2011 crop insurance ratio of claims to premiums was only 0.58 in Indiana, 0.28 in Iowa, and 0.44 in Illinois.
States hit hard by weather last year ran claim-to-premium ratios as high as 2.36 in drought-stricken Texas, 1.55 in flooded North Dakota, and 2.77 in storm-ravaged Vermont. Some regions that reported big claims last year are in relatively good shape this year.
In 2011, more than 2 million policies for various types and levels of crop insurance covered more than 265 million acres. Growers paid premiums of nearly $12 billion and insurance paid out $10.8 billion in claims.
Nearly 90% of Iowa's corn and soybean acres are covered by multiple peril crop insurance, noted William Edwards, Iowa State University extension economist, in comments this month on the Ag Decision Maker website. Of the insured acres, 90% are covered by revenue protection insurance policies.
"Yield losses will be paid at a rate equal to the average CME futures price during the month of October, if it exceeds the average February price of $5.68 for corn (December contract) or $12.55 for soybeans (November contract)," wrote Edwards.
Based on the payment guidelines, the potential claims multiply into the billions quickly.
As of July 23, FCIC reported indemnities of $547 million for 2012. Producers have filed some claims resulting from the drought, and adjustors are beginning to verify claims, says one RMA source. Claims and data go to the insurance companies, which report to RMA. Now is much too early in the season to project claims, said the RMA source. Damage levels typically vary widely across a given state, and weather can still be a big factor in crop prospects.
FCIC provides part of the reinsurance coverage for companies that write policies for growers.
American Financial Group said in its 2011 financial report that it typically reinsures 15% to 25% of its gross written premium with FCIC. Last year, it reinsured 52.5% of premiums not reinsured by the FCIC in the private market, and the company purchased stop-loss protection for the rest of its crop insurance business. AFG said it expected similar levels of reinsurance for 2012.
RMA Sets Terms, Approves Companies
RMA lists 16 companies as insurers that have signed cooperative agreements. Those agreement describe the terms under which FCIC provides reinsurance and subsidies on eligible crop insurance contracts sold by the insurance companies. To reach such an agreement with FCIC, a company must meet financial standards.
RMA sets the policy terms and conditions and establishes rates. It also revises some terms. For instance, last year RMA revised the premium rates for corn and soybeans. The agency noted that the crop insurance loss ratio had consistently been below 1.0. RMA said when it announced the corn and soybean revisions that since 1994, rising participation shifted the mix of insured growers and the loss ratio declined.
And in a July 23 bulletin, RMA administrator William Murphy said accrual of interest on unpaid spring crop insurance premium is deferred 30 days from the premium due date specified in the 2008 farm bill. For policies with a premium billing date of Aug. 15, interest on any unpaid spring crop insurance premium will not start until Nov. 1.
"Farmers and ranchers are struggling through widespread severe drought conditions in many parts of the nation, causing catastrophic damage to crops," wrote Murphy. He noted concerns about the requirement for producers to pay spring crop insurance premiums to avoid interest when those producers have not received their crop insurance indemnities. RMA also deferred collection of unpaid producer premiums from participating insurance companies until Oct. 31.
For More Information
Read more news about the 2012 drought.


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Sophia Dollars
"I'll rim your grandparents for one of these... Naahh, I'll do it for free"
"This system is the greatest currency system I've ever seen!"
Sophia Dollars, or Sophias, (symbol: ☺$) are the national currency of Uncyclopedia.
Uncyclopedia's founder was up on a hill one day staring at the city of Uncyclopedia. It was a thriving community, and the founder asked himself "This place... it's missing something. But what?" And so he thought, and mused, and puzzled about it for three days, and finally realised: There weren't any bosses yelling at employees in the city, nor banks being robbed, nor nuns getting killed for money! But the thing that brought this to the founder's attention was that no one had to pay for all the work he did for humanity! The injustice! So the founder decided to make some kind of money for people to fight over.
How It Works[edit]
Okay, so here's how the Sophias work, in handy step-by-step instructions:
- First, you need to pray to the person on the face of the dollar bill. Otherwise, the almighty goddess of Uncyclopedia In-Jokes will huff you. So say your prayers.
- After praying, do some work around Uncyclopedia. Lots of work. HARD work.
- Next, tell the person on the bill that you did work around Uncyclopedia. If you can't, proceed to tell someone else.
- Now, hope that fortune smiles on you and you get a couple of blow-jobs for your effort. If not, try again.
If you manage to earn some money, good for you!
Now give it back.
Current UnDollar Bills[edit]
Now available in an UnBank near you!