Mini-symposium on quantitative PCR
Improving the quality of the results
- February 11, 2014
- 10:00 - 12:00
- Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium (Auditorium C)
Reverse transcription (RT) is the obligatory step preceding the amplification and quantification of RNA. It is generally acknowledged as the most variable stage in the RT-qPCR protocol, with results dependent on both choice of RT enzyme and target. This perception is based largely on an analysis of enzymes carried out ten years ago, and may no longer apply to the improved enzymes and buffers in use to-day. We have analysed and compared the performance of most commercially available reverse transcriptases in terms of their sensitivity and linearity and conclude that their performance is very similar, although there are some that perform better or significantly worse than average. This talk will elaborate on our results and also discuss the potential usefulness of a non-enzymatic, hybridization-based conversion of RNA to DNA for use with more difficult templates such as those extracted from archival samples.
The MIQE guidelines describe the assay parameters to be evaluated in the lab before giving green light on a qPCR assay for use in nucleic acid quantification studies. Wet lab validation of a qPCR assay appears to be a challenge or extra burden for many users. Here, we 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 assay specificity.
Following 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 enabled 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. We will show how expression levels correlate between the different platforms and how the platform choice affects the achievable sensitivity.
Currently, a lack of consensus exists on how best to perform and interpret quantitative PCR 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.
The Minimum Information for Publication of Quantitative 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 setting up and 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. In my talk, I will focus on a few parameters of the guidelines and demonstrate how the success of your study may depend on them. I will end by summarizing a large survey of 1700 published qPCR articles assessing the compliance to the guidelines (Nature Methods, 2013).
