{'fraction_non_alphanumeric': 0.02310084406930253, 'fraction_numerical': 0.00888494002665482, 'mean_word_length': 5.119565217391305, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effects of sialic acid substitutions on recognition by Sambucus nigra agglutinin and Maackia amurensis hemagglutinin.

pseudogenes. So far, we have analyzed 10 different housekeeping genes belonging to different functional and abundance classes in various tissues and cells. We clearly observed that no gene is really constantly expressed and multiple housekeeping genes are required for an accurate normalization (Vandesompele et al., submitted for publication). One-step RT-PCR reactions are often preferred over two-step reactions, because of a presumed minimization of experimental variation, as all enzymatic steps occur in the same tube under controlled thermocycler conditions. To address the issue of reproducibility, we determined the interassay variation of our established two-step RT-PCR protocol. We have therefore subjected nine different RNA samples to two parallel rounds of DNase treatment and cDNA synthesis and quantified the relative expression levels of each of four housekeeping genes (HMBS, UBC, ACTB, and HPRT1) in all samples using the comparative Ct method. The two-step protocol is highly reproducible with Pearson correlation coefficients ranging from 0.974 to 0.988 between the expression levels of the two parallel series of cDNA samples for the four tested genes, of which one is shown in Fig. 3. The interassay variation was calculated as the median coefficient of variation (standard deviation divided by the arithmetic mean) for the paired relative quantities obtained in the two parallel series for the four genes and amount to 7.7%. This figure is only slightly higher than the observed median intraassay variation of 5.8% (reflecting the variation between duplicated reactions in the same PCR run; based on 25 different genes tested on 30 samples, data not shown) and demonstrates the reproducibility of the two-step approach. To summarize, SYBR green I is the detection format of choice for accurate and reproducible real-time transcript abundance measurements of a large series of genes. Obscuring PD observed in one-step RT-PCR reactions were eliminated by the use of a two-step and DNase treatment of RNA prior to cDNA synthesis was shown to be a prerequisite for accurate RT-PCR and facilitated primer design.