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.
Highlights
Reverse Transcription (RT) - quantitative Polymerase Chain Reaction has become a versatile technique to examine expression changes of one or more genes of interest in various pathological states
Publications were evaluated according to the year of publication, the type and amount of samples, the number of reference genes, the methods used to determine RNA integrity, the amount of total RNA initially utilized for RT and/or quantitative Polymerase Chain Reaction (qPCR), the number of replicates in qPCR, the details on serial dilutions for calibration curve and efficiency, the utilized qPCR chemistry (SYBRgreen or TaqMan), and the mathematical approaches for reference gene expression stability measurement
To study the acceptance of MIQE in more detail, we performed a literature review of publications proposing a set of putative reference genes (n$5) in human samples for RT-qPCR
Summary
Reverse Transcription (RT) - quantitative Polymerase Chain Reaction (qPCR) has become a versatile technique to examine expression changes of one or more genes of interest in various pathological states. RT-qPCR is a robust assay that uses wellestablished chemistry and data analysis and is superior to techniques such as Southern blotting or DNA sequencing [2]. There are inconsistencies in the use of total RNA extraction methods, RNA quantity per reaction [3], RNA integrity assessments [4], qPCR master mixes and in the various manufacturers of the reverse transcription kits. The use of different qPCR detection methods such as dye- or probe-based systems broadens the spectrum of potential applications. The application of these guidelines will deliver better and reproducible results by reporting parameters such as RNA integrity, reaction volume, cDNA/RNA concentration, or calibration curves [6]
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