Volume-Related Inhibitors Standardization for Reverse Transcription Quantitative Polymerase Chain Reaction Experiments
Poster Apr 24, 2012
Pascal Pugniere, Sebastien Banzet, Thomas Chaillou, Catherine Mouret and Endre Peinnequin
A large part of the reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) data depends on technical variations. Such variations are mainly attributable to the RT step. Standardization is not always possible, and compromises must be found. Due to technical requirements, the current consensus is that a constant amount of total RNA should be used for the RT step (CA-RT). Because RNA isolation yields are variable, variable volumes of nucleic acid extracts are used in RT Reaction. To overcome such drawbacks, we proposed to carry out the RT reaction with constant volume of RNA extract by preserving a constant RNA amount through the supplementation of yeast transfer RNA (CV-RT). The aim of this study was to determine whether CV-RT could improve the reliability of RT-qPCR assessment compared with the classical use of a constant amount of total RNA (CA-RT).
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE