oru.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Pitfalls of reverse transcription quantitative polymerase chain reaction standardization: Volume-related inhibitors of reverse transcription
Genomic Core Facility, IRBA La Tronche, La Tronche, France.
Operational Environments, IRBA La Tronche, La Tronche, France.
Operational Environments, IRBA La Tronche, La Tronche, France.ORCID iD: 0000-0002-5322-4150
Genomic Core Facility, IRBA La Tronche, La Tronche, France.
Show others and affiliations
2011 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 415, no 2, p. 151-157Article in journal (Refereed) Published
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 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.

Place, publisher, year, edition, pages
Academic Press, 2011. Vol. 415, no 2, p. 151-157
Keyword [en]
Reverse transcription; RT-qPCR; mRNA quantification; Standardization; Inhibitor; MIQE guidelines
National Category
Analytical Chemistry Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:oru:diva-66017DOI: 10.1016/j.ab.2011.04.008ISI: 000291904700008PubMedID: 21530480Scopus ID: 2-s2.0-79958283994OAI: oai:DiVA.org:oru-66017DiVA, id: diva2:1192517
Note

Funding Agency:

Delegation Generale pour l'Armement

Note: Institut de recherche biomédicale des armées (IRBA)

Available from: 2018-03-22 Created: 2018-03-22 Last updated: 2018-05-08Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMedScopus

Authority records BETA

Chaillou, Thomas

Search in DiVA

By author/editor
Chaillou, Thomas
In the same journal
Analytical Biochemistry
Analytical ChemistryBiochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 19 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf