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Jan Hellemans, Pieter Mestdagh, Barbara D’haene, Ariane Deganck, Jo Vandesompele Biogazelle, Belgium |
Abstract
The MIQE guidelines describe the assay parameters to be evaluated in the lab before giving green light on a qPCR assay for use in quantification studies. Wet lab validation of a qPCR assay appears to be a challenge for some users, or an unwanted extra burden for many others. We will describe the procedures by which we have successfully completed the full wet-lab validation of RT-qPCR assays for all protein coding genes of the human and mouse transcriptome. One of the key improvements in our validation approach is the use of massively parallel amplicon sequencing for an unprecedented stringency on the assay specificity.
After extensive validation, we have used all human assays (n=18,841) on the four MicroArray Quality Control (MAQC) RNA samples to obtain the first RT-qPCR based human transcriptome expression profile. The data reveal an impressive dynamic range with 90% of protein coding genes detected over a 20 million fold range. In addition, this study enables a thorough comparison of various quantification performance aspects of qPCR with those of microarrays and RNA sequencing obtained in the MAQC and SEQC studies, respectively. Finally, this study yields insights in the usefulness of the qPCR Reference RNA sample as positive control and another view on the publicly available data of the genes expressed in brain.
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