Abstract
Proper normalization of RT-qPCR data is pivotal to the interpretation of results and accuracy of scientific conclusions. Though different approaches may be taken, normalization against multiple reference genes is now standard practice. Genes traditionally used and deemed constitutively expressed have demonstrated variability in expression under different experimental conditions, necessitating the proper validation of reference genes prior to utilization. Considering the wide use of fibroblasts in research and scientific applications, it is imperative that suitable reference genes for fibroblasts of different animal origins and conditions be elucidated. Previous studies on bovine fibroblasts have tested limited genes and/or samples. Herein, we present an extensive study investigating the expression stability of 16 candidate reference genes across 7 untreated bovine fibroblast cell lines subjected to controlled conditions. Data were analysed using various statistical tools and algorithms, including geNorm, NormFinder, BestKeeper, and RefFinder. A combined use of GUSB and RPL13A was determined to be the best approach for data normalization in untreated bovine fibroblasts.
Highlights
Proper normalization of Reverse transcription (RT)-qPCR data is pivotal to the interpretation of results and accuracy of scientific conclusions
The most commonly used “classical” reference gene (RG), including β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine–guanine phosphoribosyl transferase (HPRT) and 18S ribosomal RNA (18S rRNA), are carryovers from references used in Northern blotting, RNase protection and conventional RT-PCR assays, which were suitable for these non- and semi-quantitative techniques where qualitative changes were evaluated[20]
M values for all genes fell within range for inclusion (M < 0.5), B2M, HPRT1, and RAD50 had the lowest stability while GUSB, RPL13A, and Scientific Reports | (2021) 11:10253 |
Summary
Proper normalization of RT-qPCR data is pivotal to the interpretation of results and accuracy of scientific conclusions. Fibroblasts have been used as donor cells in somatic cell nuclear transfer (SCNT)[6,7], and induced pluripotent stem cell applications (iPSC)[8,9,10], which present powerful tools for disease modeling in vitro[11,12,13,14,15,16], personalized and regenerative medicine, oncogenic applications[17], and wildlife conservation[1], among others In many of these studies, describing transcriptional changes of key regulatory genes within the fibroblast has been crucial to the complete understanding of the cellular mechanisms underpinning their function. Electron transporter in TCA cycle and respiratory chain NM_174178.2 pre-mRNA splicing; as a component of pre-catalytic spliceosome "B" complexes
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