title: Pitfalls of reverse transcription quantitative polymerase chain reaction standardization: Volume-related inhibitors of reverse transcription.
abstract: 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 reverse transcription step. Standardization is a key factor in decreasing the intersample variability. However, an ideal 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, such a practice requires the use of variable volumes of nucleic acid extracts in RT reaction. We demonstrate that some RNA extracts contain both exogenous and endogenous inhibitors. These inhibitors induce a decrease in RT efficiency that significantly impairs the reliability of RT-qPCR data. Conversely, these inhibitors have a slight effect on the qPCR step. To overcome such drawbacks, we proposed to carry out the RT reaction with a constant volume of RNA extract by preserving a constant RNA amount through the supplementation of yeast transfer RNA (CV-RT). We show that CV-RT, compared with the usual CA-RT, allows us to decrease the RT-qPCR variability induced by intersample differences. Such a decrease is a prerequisite for the reliability of messenger RNA quantification.
authors.affiliation: Genomic Core Facility, IRBA La Tronche, 38702 La Tronche, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Pugniere
authors.forename: Sebastien
authors.initials: S
authors.lastname: Banzet
authors.forename: Thomas
authors.initials: T
authors.lastname: Chaillou
authors.forename: Catherine
authors.initials: C
authors.lastname: Mouret
authors.forename: Andre
authors.initials: A
authors.lastname: Peinnequin
mesh_terms: D012313:RNA / Q000032:analysis / Q000037:antagonists & inhibitors / Q000592:standards; D012333:RNA, Messenger / Q000032:analysis* / Q000592:standards; D012343:RNA, Transfer / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*
chemical_list: D012333:RNA, Messenger; D018894:Reverse Transcriptase Inhibitors; D012313:RNA; D012343:RNA, Transfer; D012194:RNA-Directed DNA Polymerase