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High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.


Introduction
Types I and III interferons (IFN) are critical in the immune response to viruses and other pathogenic stimuli and present in all vertebrates 1 .
Immune and non-immune cells express and secrete, as well as respond to, IFN 2 . Innate immune sensors, such as toll-like receptors (TLR), STING, and RIG-I, induce type I and III IFN expression upon detection of pathogen associated molecular patterns (PAMP) 3,4 . In humans, type I IFN include IFN-β, -ω, -κ, and 12 subtypes of IFN-α, and bind to the IFNAR1/IFNAR2 receptor complex 2,5 . Type III IFN include IFN-λ1, -λ2, and -λ3 and bind to the IL10RB/IL28RA receptor complex 2 . Classically, types I and III IFN bind to their respective receptor complexes which then recruit STAT1/STAT2 heterodimers and initiate transcription of interferon stimulated genes (ISG) 6 . ISG are involved in a diverse range of functions, from antiviral and antiproliferative activity to activation of the adaptive immune response 7 .
The numerous mechanisms pathogens have evolved to evade, subvert, and hijack elements of the IFN signaling pathway demonstrate the importance of IFN in the innate immune response 8 . For example, vaccinia virus expresses a decoy receptor with IFNAR1 homology that sequesters type I IFN 9 while a Yaba-like disease virus secretes a glycoprotein that inhibits type I and III IFN proteins 10 . In addition to their role in host defense, IFN are also implicated in cancer surveillance and a number of auto-inflammatory diseases: silencing of IFN expression in breast cancer cells restricts immunosurveillance 11 , overproduction of IFN-α is a mechanism in the development of systemic lupus erythematosus 12 , and errant activation of STING leads to systemic inflammation caused by excessive amounts of IFN in STING-associated vasculopathy 13 . Therapeutically, IFN are used to treat multiple sclerosis 14 , chronic viral infections such as HBV 15 and HCV 16,17 , and cancers such as hairy cell leukemia 18 and chronic myelogenous leukemia 19 . Questions about the relevance of IFN in a particular physiological process continuously reveal the ubiquitous nature of this cytokine family.
Type I IFN, especially the IFN-α subtypes, are often considered as one entity [20][21][22][23] , rather than as a group of closely related, but distinct, proteins. The existence and persistence of multiple IFN species including the IFN-α subtypes, throughout vertebrate evolution 24 suggests that at least a subset of these subtypes have specific or unique functions. It is possible that defining patterns of IFN expression will decipher and help characterize the specific functions of one or more of the subtypes 17 . The challenge of studying the type I and III IFN subtypes is based on their shared sequence identity: the twelve IFN-α subtypes share >50% 25 and the IFN-λ subtypes share 81-96% 26 of their amino acid sequences.
In the described qRT-PCR assay, molecular beacon (MB) and locked nucleic acid (LNA) fluorescent probes discriminate single base pair differences between the highly similar IFN subtype sequences and allow for the characterization of the IFN expression signature. The assay's 384-well plate format includes both quantitative (transcript standards) and semi-quantitative (the housekeeping genes (HKG)) measures, allowing for analysis by transcript copy number and ∆Cq respectively. Batch assembly, facilitated by automated multichannel pipetting, and long term storage, possible through drying the primer/probe (Pr/Pb) sets on the plates, enhance the reproducibility, utility, and practicality of the assay.
2. Place a new 96-well plate in a chilled 96-well cooling block and designate the wells for each Pr/Pb set or NTC mix the wells receive (see Figure 2 5. Seal the 96-well plate with an adhesive plate seal and centrifuge for 1 min at 700 x g to ensure the contents are at the bottom of the wells. 6. Place the 96-well plate in the vortex mixer using a 96-well plate adaptor and mix for 1 min at 1,000 rpm. Centrifuge the 96-well plate for 5 min at 700 x g. 7. Store the 96-well source plate in the dark at 4 °C if using in the same day, otherwise store at -20 °C until use.
2. Prepare the 384-well assay plates by adding the Pr/Pb sets using the automated multichannel pipettor.
NOTE: Preparation of six assay plates from the 96-well source plate will take 3-4 hr. 5. Centrifuge the plate for 1 min at 700 x g. Remove the adhesive plate seal, place the 384-well assay plate into the plate dryer and position the 384-well manifold directly above the wells. 6. When the 384-well assay plate's contents dry, apply a new adhesive plate seal, wrap in foil, label, and store in the dark at 4 °C until use. NOTE: Plates can be stored at 4 °C for at least 6 months. 7. Repeat steps 3.2.3-3.2.6 until 6 x 384-well assay plates are prepared or the liquid in the 96-well source plate is depleted. 8. Turn off the cooling nest, close the software, and switch off the automated multichannel pipettor. Turn off the plate dryer.

Loading and Running a 384-well Assay Plate
1. Prepare two housekeeping gene (HKG) well mixes, which consist of the Pr/Pb set for a HKG, SSDNA, PCR master mix, and water, to be added to a dried 384-well assay plate ( Figure 3D). NOTE: Preparation of the mixes and loading the assay plate will take 1-2 hr. Running the assay plate will take less than 2 hr. 1. Label a 1.5 ml tube for each HKG mix. 2. Dilute 2 µl of SSDNA (10 mg/ml) with 84.9 µl water. Add 2 µl of the diluted SSDNA, 11.8 µl of water, 23 µl of master mix, and 9.2 µl of the correct 20x HKG Pr/Pr set to each tube, vortex, and briefly centrifuge. 3. Use glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin C (UBC) as the HKG for the human assay (see Materials  Table). Use GAPDH and 18S ribosomal RNA as the HKG for the rhesus assay. NOTE: The HKG used to perform the assay are flexible and should reflect the genes appropriate for the cell type being tested. GAPDH, UBC, and 18S are examples of commonly used HKG; other HKG may be more appropriate.
2. Prepare the sample and positive control well mixes, which consist of the sample or positive control cDNA, master mix, and water, to be added to a dried 384-well assay plate. Prepare the sample and the positive control mixes in enough quantity to dispense to 21 plate wells ( Figure  3D). 3. Prepare the standards and NTC well mixes, consisting of master mix and water, to be added to a dried 384-well assay plate ( Figure 3D). For the standards well mix, add 165 µl of water to 275 µl of master mix in a 1.5 ml tube. For the NTC well mix, add 52.5 µl of water to 52.5 µl of master mix in a 1.5 ml tube. Vortex and briefly centrifuge the tubes. 4. Prepare a dried 384-well assay plate for loading of the mixes and samples.
1. Remove a dried 384-well assay plate from 4 °C and centrifuge for 1 min at 700 x g. 2. Place the plate on a chilled 384-well cooling block, remove the adhesive plate seal and outline the wells of the plate to designate where the various mixes and samples will be pipetted (Figure 1).

5.
Load the 384-well assay plate with standard well mix, standards, NTC well mix, samples, positive control, and HKG mix ( Figure 3E). Vortex and briefly centrifuge all solutions before dispensing to the plate. 3. Fill any empty wells with 7.5 µl of the leftover water and master mix mixture with the 30 µl electronic multichannel pipette; 7.5 µl of water alone will also work. 4. Seal the 384-well assay plate with the optical adhesive film and centrifuge for 1 min at 700 x g. Vortex the sealed 384-well plate in the vortex mixer for 2 min at 2,600 rpm and centrifuge for 5 min at 700 x g. 5. Place the sealed 384-well assay plate in the qRT-PCR machine, open the template for the assay layout and begin the run. NOTE: Results can be exported as a spreadsheet or as a text file for analysis. 6. Use the following optimal thermal cycler reaction conditions for the assay: i) 50 °C for 2 min, ii) 95 °C for 10 min, iii) 95 °C for 25 s, iv) 59 °C for 1 min. Repeat steps iii and iv for 40 cycles. 7. Export the raw data from the qRT-PCR platform into a spreadsheet application software. Plot each 4 point standard set as a standard curve to observe linearity. Perform analysis by calculating the ∆Cq or by copy number based on the four point standard curves 27 .

Representative Results
The qRT-PCR assay described can be implemented to analyze expression patterns of types I and III IFN in a variety of cell types and contexts. For example, human type I and III IFN expression signatures were analyzed in peripheral blood mononuclear cells (PBMC) from 6 donors stimulated with TLR ligands; poly I:C (25 μg/ml), LPS (10 ng/ml), imiquimod (10 μM), and CpG oligonucleotides (1 μM) (Figure 4). Data were analyzed using a spreadsheet application software and presented as radar charts with the IFN-α subtypes arranged clockwise according to the phylogenetic tree of their protein sequence 27 . The radar charts of human IFN expression are presented in a log scale calculated using the two different methods of analysis incorporated into the assay design: absolute Cq value normalized to HKG (ΔCq), and copy number of template per microgram (μg) of total RNA. Copy number values are calculated from the results of a transcript's standard curve. The data shows that human IFN expression signatures elicited by TLR agonists are ligand specific.

See Figure 4 Below
As demonstrated for the human IFN expression signatures, expression signatures of types I and III IFN in rhesus macaques were also TLR ligand specific. PBMC from 3 donors were stimulated with poly I:C (50 μg/ml), LPS (10 μg/ml), and imiquimod (10 μg/ml) for 3 hr (Figure 5). IFN subtype expression in unstimulated cells at baseline was low. A limited number of IFN-subtypes were expressed in response to LPS and poly I:C. In contrast, IFN expression in response to imiquimod was high and the subtype expression was broad. Expression of IFN-β and IFN-λ1 was enhanced by all three TLR agonists 28 .

Discussion
This report describes design, batch production, and an approach towards analysis of an assay to measure transcription of a set of highly similar genes in a research laboratory setting. The high-throughput qRT-PCR assay reported here measures the IFN-and -λ subtypes with high specificity. This method involves two key aspects, the design of Pr/Pb sets that discriminate between members of a homologous gene family and the development of a production platform for the creation of reliable and consistent 384-well assay plates preloaded with the Pr/Pb sets. The qRT-PCR probes incorporate a structural (MB) or chemical (LNA) approach towards enhancing their specificity 29 . For two of the primer sets in the rhesus assay ( Table 2), the amplification-refractory mutation system (ARMS) was incorporated into the primer sequences to further enhance specificity 30 . While it is generally best to target exon-exon junctions to enhance specificity of a qRT-PCR reaction, this was not possible because the type I IFN genes lack introns. Therefore, genomic DNA will be amplified in the PCR reaction, and must be degraded by DNase treatment after RNA extraction.
The target region for the Pr/Pb sets was restricted to the coding regions of the mature peptide for each IFN. Because of the high sequence similarity among the IFN-α subtypes, particularly the mature peptide region, it was sometimes necessary to compromise sensitivity to ensure specificity. This was particularly the case with IFN-α17, where the mature peptide transcript has only four unique bases when compared against the other IFN-α subtypes. Targeting IFN-α17 required primers that bind the transcripts of multiple subtypes, restricting specificity to the probe. As a consequence, the PCR reaction will amplify subtypes other than IFN-α17, thereby consuming a substantial percentage of the PCR reagents and lowering the amplitude of the fluorescence signal from the specific probe for IFN-α17. An additional challenge towards designing sensitive and specific Pr/Pb sets for highly similar genes such as the IFN is the possibility that the annotated sequences in the GenBank database may not be complete or comprehensive at time of design. Again, this was a challenge for IFN-α17, in which newly annotated sequences do not perfectly align with the version of the sequence in the database at the time of design. Therefore, when designing Pr/Pb sets for genes that have not been intensively studied, it is wise to periodically check the latest annotated sequence of a target gene. Finally, it is necessary to ensure that the Pr/Pb sets do not amplify pseudogenes that may be transcribed but not translated.
After designing the Pr/Pb sets, the next challenge is optimizing the PCR conditions of seventeen different Pr/Pb sets on one 384-well plate. Transcript standards are important in testing the specificity of a Pr/Pb set and become essential for harmonizing the qRT-PCR conditions of the numerous different PCR reactions. Testing a Pr/Pb set against the transcript standards establishes its efficiency and sensitivity; plasmids that express highly similar pseudogenes may be necessary to ensure that the Pr/Pb set selectively measures transcription of the functional gene. The transcript standards also provide a quantitative means of analysis (number of transcripts), in addition to the semi-quantitative analysis relative to a housekeeping gene (ΔCq).
Robotic multichannel pipetting from a 96-well source plate into multiple 384-well assay plates improves the precision and consistency of interplate results. Salmon sperm DNA (SSDNA) is used as a carrier that stabilizes and preserves the Pr/Pb sets for long term storage, as does drying the reagents dispensed into the plates. Drying the plates also decreases the volume of the reaction necessary for reproducible results, which in turn preserves precious samples and decreases the use of costly reagents. Through these steps, batches of plates are assembled that provide precision and consistency for more than six months.
Following plate preparation, quality control measures are critical to check the consistency of a batch of plates. For this purpose, an additional four sets of standards are run on a plate. The "5x standard" plate tracks performance and creates a data set to which an individual plate's standards are compared. While ten 10-fold dilutions of each standard are used during assay design, space considerations require that four points are used for the standard curve on each assay plate, and for the 5x standard plate. Additionally, a positive control should be included on each plate to ensure the validity of the plate.
Typically, it takes 3-4 hr to prepare six assay plates from one Pr/Pb source plate; it is feasible to prepare twelve plates in a single day in a research laboratory. Since each human IFN subtype assay plate examines seventeen Pr/Pb sets and can accommodate fifteen experimental samples, one day of assembly produces a batch of plates with the capacity to generate up to 3,060 experimental data points. Raw data from the qRT-PCR platform can be processed and assembled using programming scripts in a spreadsheet application software to automatically fill a predesigned analysis template. This method minimizes hands-on data entry, thereby preventing copying errors and allows the investigator to focus on data analysis rather than data assembly. As described here, this high-throughput qRT-PCR assay can be applied to measure the expression of interferon subtypes in human or rhesus macaque samples and could be adapted to use for other species or homologous gene sets. The flexibility of the plate layout allows the user to change primer/probe sets to tailor the genes of interest toward a particular cell type or model system. This assay can be applied to measure IFN expression signatures in cell culture models studying pathogens or in patient samples in the context of disease models to elucidate the signaling mechanisms involved in immune response.

Disclosures
VPM and RLR are co-inventors of the technology to measure expression of IFN-and IFN-subtypes and derive royalty income from it. The remaining authors declare no conflict of interest.