Control selection for RNA quantitation.

By Toshihide Suzuki, P. Higgins, D. Crawford. BioTechniques, 2000

921 citations recorded by the source corpus.

The study of mammalian gene expression is often carried out at the level of mRNA. In such analyses, one usually measures the amount of an mRNA of interest under different conditions such as stress, growth, development, cell and tissue localization or as part of an evaluation of the effects of gene transfection. A variety of techniques exist to measure gene expression and most commonly involve Northern hybridization analysis, ribonuclease protection or RT-PCR. Common to all of these assays is the inclusion of a so-called loading or internal control (i.e., analysis of an mRNA that does not change in relative abundance during the course of treatments). Here, we discuss the uses and pitfalls of the most popular of these controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, with special emphasis on precautions associated with the use of GAPDH.

Subfields: Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/886533cc3f3d30b36cff685df182d251f089b722

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Citations in the corpus

Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations (2004, methodology): “…parameters of the quantification procedure (e.g. inhibitory factors of the tissue, integrity of the RNA, loading error, enzyme or primer performance, etc.) must be controlled, numerous standardisation methods have been proposed (Suzuki et al. 2000, Thellin et al. 1999, Vandesompele et al. 2002).”
Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon (2005, background): “Recently, the use of these two genes as endogenous controls has been scrutinized, and several studies have documented that the GAPDH and β-actin genes should be used with caution as controls [2,8,9].”
Guideline to reference gene selection for quantitative real-time PCR. (2004, background): “[4] described that in 1999 over 90% of the RNA transcription analyses published in high impact journals, used only one reference gene.”
Comprehensive Algorithm for Quantitative Real-Time Polymerase Chain Reaction (2005, methodology): “We used the geometric mean of three control genes (Actin, G3PDH, and 18S rRNA) for accurate normalization (Vandesompele et al., 2002) to minimize errors from potential variance in a single control gene (Schmittgen and Zakrajsek, 2000; Suzuki et al., 2000).”
Evidence Based Selection of Housekeeping Genes (2007, methodology): “GAPDH and ACTB were used as single control genes in more then 90% of the cases in high impact journals.[11] Commonly used control genes are historical carryovers and were considered good references for many years in techniques where a qualitative change was being measured, because these genes are ex”
Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. (2002, background): “Ideally sample-to-sample variation could be corrected by measuring simultaneously the levels of a single universal cellular RNA that is present at constant levels during all stages of development, regardless of tissue or experimental treatment [4].”
Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1[w] (2005, background): “Unfortunately, the transcript levels of these reference genes are not always stable (Thellin et al., 1999; Suzuki et al., 2000; Lee et al., 2001; this study), and their use for normalization purposes can compromise gene expression studies.”
Reference genes in real-time PCR (2013, background): “That this is the wrong approach is confirmed by practice for several years now, but the beginnings were tough as presented by Suzuki et al. (2000); it was the case for more than 90 % of the articles concerning the analysis of RNA transcript that were published in 1999 in high impact journals.”
Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. (2003, background): “The comparable levels of b-actin expression given equivalent RNA loading demonstrate that this gene is a suitable internal control for normalising RNA loading in this experimental context (17,18).”
Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. (2006, background): “However, several reports demonstrate that the transcript levels of these genes also vary considerably under different experimental conditions [10–13] and are consequently unsuitable for gene expression studies.”
Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. (2002, methodology): “I would like to thank Qiagen for their gift of Sensiscript, Biosearch for their gift of IGF-II probes, Adrian Palmer (Roche) for carrying out the extraction of RNA on the Magnapure and the RT-PCR on the Lightcycler and Jane Theaker and Stephen Little for additional information on Scorpions.”
CB2: a cannabinoid receptor with an identity crisis (2010, background): “Their high sensitivity makes their interpretation subject to several considerations (Suzuki et al., 2000; Lion, 2001).”