Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.

Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since im...

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Autores principales:	Francis Jacob, Rea Guertler, Stephanie Naim, Sheri Nixdorf, André Fedier, Neville F Hacker, Viola Heinzelmann-Schwarz
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f424f0a7bead46caa47d6a4e8a4e5915
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spelling	oai:doaj.org-article:f424f0a7bead46caa47d6a4e8a4e59152021-11-18T07:53:14ZCareful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.1932-620310.1371/journal.pone.0059180https://doaj.org/article/f424f0a7bead46caa47d6a4e8a4e59152013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23554992/?tool=EBIhttps://doaj.org/toc/1932-6203Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; 2) compare various algorithms in their expression stability; 3) identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines. A PubMed database review was performed and publications since 2009 were selected. Twelve putative reference genes were profiled in normal and various cancer cell lines (n = 25) using 2-step RT-qPCR. Investigated reference genes were ranked according to their expression stability by five algorithms (geNorm, Normfinder, BestKeeper, comparative ΔCt, and RefFinder). Our review revealed 37 publications, with two thirds patient samples and one third cell lines. qPCR efficiency was given in 68.4% of all publications, but only 28.9% of all studies provided RNA/cDNA amount and standard curves. GeNorm and Normfinder algorithms were used in 60.5% in combination. In our selection of 25 cancer cell lines, we identified HSPCB, RRN18S, and RPS13 as the most stable expressed reference genes. In the subset of ovarian cancer cell lines, the reference genes were PPIA, RPS13 and SDHA, clearly demonstrating the necessity to select genes depending on the research focus. Moreover, a cohort of at least three suitable reference genes needs to be established in advance to the experiments, according to the guidelines. For establishing a set of reference genes for gene normalization we recommend the use of ideally three reference genes selected by at least three stability algorithms. The unfortunate lack of compliance to the MIQE guidelines reflects that these need to be further established in the research community.Francis JacobRea GuertlerStephanie NaimSheri NixdorfAndré FedierNeville F HackerViola Heinzelmann-SchwarzPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 3, p e59180 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Francis Jacob Rea Guertler Stephanie Naim Sheri Nixdorf André Fedier Neville F Hacker Viola Heinzelmann-Schwarz Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.
description	Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; 2) compare various algorithms in their expression stability; 3) identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines. A PubMed database review was performed and publications since 2009 were selected. Twelve putative reference genes were profiled in normal and various cancer cell lines (n = 25) using 2-step RT-qPCR. Investigated reference genes were ranked according to their expression stability by five algorithms (geNorm, Normfinder, BestKeeper, comparative ΔCt, and RefFinder). Our review revealed 37 publications, with two thirds patient samples and one third cell lines. qPCR efficiency was given in 68.4% of all publications, but only 28.9% of all studies provided RNA/cDNA amount and standard curves. GeNorm and Normfinder algorithms were used in 60.5% in combination. In our selection of 25 cancer cell lines, we identified HSPCB, RRN18S, and RPS13 as the most stable expressed reference genes. In the subset of ovarian cancer cell lines, the reference genes were PPIA, RPS13 and SDHA, clearly demonstrating the necessity to select genes depending on the research focus. Moreover, a cohort of at least three suitable reference genes needs to be established in advance to the experiments, according to the guidelines. For establishing a set of reference genes for gene normalization we recommend the use of ideally three reference genes selected by at least three stability algorithms. The unfortunate lack of compliance to the MIQE guidelines reflects that these need to be further established in the research community.
format	article
author	Francis Jacob Rea Guertler Stephanie Naim Sheri Nixdorf André Fedier Neville F Hacker Viola Heinzelmann-Schwarz
author_facet	Francis Jacob Rea Guertler Stephanie Naim Sheri Nixdorf André Fedier Neville F Hacker Viola Heinzelmann-Schwarz
author_sort	Francis Jacob
title	Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.
title_short	Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.
title_full	Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.
title_fullStr	Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.
title_full_unstemmed	Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.
title_sort	careful selection of reference genes is required for reliable performance of rt-qpcr in human normal and cancer cell lines.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/f424f0a7bead46caa47d6a4e8a4e5915
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Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.

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