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Volume-related inhibitors standardization for reverse transcription quantitative polymerase chain reaction experiments
University of Kentucky, Lexington KY, United States .ORCID iD: 0000-0002-5322-4150
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2012 (English)Conference paper, Poster (with or without abstract) (Refereed)
Abstract [en]

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 decreased RT efficiency that significantly impairs the reliability of RT-qPCR data. Conversely, these inhibitors have slight effects 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.

Place, publisher, year, edition, pages
2012.
National Category
Biochemistry and Molecular Biology Analytical Chemistry
Identifiers
URN: urn:nbn:se:oru:diva-66331OAI: oai:DiVA.org:oru-66331DiVA, id: diva2:1194902
Conference
Congress of Genomics Research, Boston, USA, April 19-20, 2012
Available from: 2018-04-04 Created: 2018-04-04 Last updated: 2018-05-15Bibliographically approved

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Chaillou, Thomas

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