Abstract
BackgroundQuantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantifying mRNA abundance. With relative expression analysis, however, reliable data output is dependent on stably expressed reference genes across the samples being studied. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes.PurposeThis study was aimed to investigate the optimal reference genes for PCR in ARM rat models.MethodsWe selected 15 commonly used reference genes (Rps18, Actb, B2m, Gapdh, Ppia, Hprt1, Pgk1, Ywhaz, Tbp, Ubc, Rps16, Rpl13a, Rplp1, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. The expression stability and variability of these transcripts were subsequently evaluated using four methods (geNorm, NormFinder, comparative ΔCt, and BestKeeper).ResultsThe abundance of the candidate reference genes was qualified by RT-qPCR and the cycle threshold (Ct) values ranged between 14.07 (Rplp1) and 21.89 (Sdha). In the overall candidate genes, different variations existed across the different algorithms. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps18, Sdha, and Hmbs.ConclusionsThe most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps18 in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses.
Highlights
Anorectal malformations (ARMs) are common congenital gastrointestinal malformations manifested by anal stenosis, ectopic anus, and urethrorectal fistula (Bai et al, 2004; De Blaauw et al, 2013; Wijers et al, 2014)
Many factors can affect the results of PCR, including the selection of the reference genes (Ferguson et al, 2010; Guo et al, 2013)
An ideal reference gene should be stably expressed in all the samples without being affected by species, tissues, and development (Harris, Reeves & Phillips, 2009; Mughal et al, 2018)
Summary
Anorectal malformations (ARMs) are common congenital gastrointestinal malformations manifested by anal stenosis, ectopic anus, and urethrorectal fistula (Bai et al, 2004; De Blaauw et al, 2013; Wijers et al, 2014). Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes. Purpose: This study was aimed to investigate the optimal reference genes for PCR in ARM rat models. Methods: We selected 15 commonly used reference genes (Rps, Actb, B2m, Gapdh, Ppia, Hprt, Pgk, Ywhaz, Tbp, Ubc, Rps, Rpl13a, Rplp, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps, Sdha, and Hmbs. Conclusions: The most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses
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