Abstract
In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA.
Highlights
Despite the advent of high-throughput methods such as RNA-sequencing to measure transcript abundance in cells and tissues, quantitative RT-PCR (RT-qPCR) remains the method of choice for many, when only a selected number of genes are to be analysed
One group was exposed perinatally to a mixture of 13 known endocrine disrupting compounds at a dose estimated at 450-times higher than that of human exposure, designated Mix450 (Christiansen et al, 2012), with juvenile tissue samples collected on postnatal days (P): livers on P13; testis, prostate and adrenal on P16; ovaries on P17; and adult tissues after P55
A second and third group of juvenile male rats was exposed to 5 mg/kg perfluoronanoic acid (PFNA) and 5 mg/kg PFNA in addition to a mixture of 14 chemicals (PFNA/mix), respectively (Hadrup et al, 2015), and tissues were collected from adult rats
Summary
Despite the advent of high-throughput methods such as RNA-sequencing to measure transcript abundance in cells and tissues, quantitative RT-PCR (RT-qPCR) remains the method of choice for many, when only a selected number of genes are to be analysed. A growing body of evidence has clearly shown this not to be the case and that ‘stably expressed’ reference genes must be determined for each cell/tissue type during different stages of development and under all experimental conditions (reviewed by Huggett et al, 2005; Kozera & Rapacz, 2013). This can quickly become a Herculean task and likely a major reason why it remains so frequently overlooked. Piller and co-workers scrutinized recent publications in their field of research—rat spared nerve injury model of neuropathic pain—for evidence-based use of reference genes in RT-qPCR experiments and found that only 2 out of 26 peer-reviewed articles referred to proper validation for their reference genes (Piller, Decosterd & Sutler, 2013)
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