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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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Listed:
  • Francis Jacob
  • Rea Guertler
  • Stephanie Naim
  • Sheri Nixdorf
  • André Fedier
  • Neville F Hacker
  • Viola Heinzelmann-Schwarz

Abstract

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.

Suggested Citation

  • Francis Jacob & Rea Guertler & Stephanie Naim & Sheri Nixdorf & André Fedier & Neville F Hacker & Viola Heinzelmann-Schwarz, 2013. "Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines," PLOS ONE, Public Library of Science, vol. 8(3), pages 1-8, March.
  • Handle: RePEc:plo:pone00:0059180
    DOI: 10.1371/journal.pone.0059180
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    1. Marcelo T Moura & Roberta L O Silva & Pábola S Nascimento & José C Ferreira-Silva & Ludymila F Cantanhêde & Ederson A Kido & Ana M Benko-Iseppon & Marcos A L Oliveira, 2019. "Inter-genus gene expression analysis in livestock fibroblasts using reference gene validation based upon a multi-species primer set," PLOS ONE, Public Library of Science, vol. 14(8), pages 1-20, August.
    2. Nirmal Kumar Sampathkumar & Venkat Krishnan Sundaram & Prakroothi S Danthi & Rasha Barakat & Shiden Solomon & Mrityunjoy Mondal & Ivo Carre & Tatiana El Jalkh & Aïda Padilla-Ferrer & Julien Grenier & , 2022. "RNA-Seq is not required to determine stable reference genes for qPCR normalization," PLOS Computational Biology, Public Library of Science, vol. 18(2), pages 1-24, February.
    3. Jose V Die & Ransom L Baldwin & Lisa J Rowland & Robert Li & Sunghee Oh & Congjun Li & Erin E Connor & Maria-Jose Ranilla, 2017. "Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium," PLOS ONE, Public Library of Science, vol. 12(2), pages 1-13, February.

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