Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations

Caiyun Long; Yunxia Xiao; Siying Li; Xiaobing Tang; Zhengwei Yuan; Yuzuo Bai

doi:10.7717/peerj.6829

Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations

PeerJ. 2019 Apr 23:7:e6829. doi: 10.7717/peerj.6829. eCollection 2019.

Authors

Caiyun Long¹, Yunxia Xiao¹, Siying Li¹, Xiaobing Tang¹, Zhengwei Yuan², Yuzuo Bai¹

Affiliations

¹ Department of Pediatric Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China.
² The Key Laboratory of Health Ministry for Congenital Malformation, Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China.

Abstract

Background: Quantitative 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.

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 (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).

Results: The 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.

Conclusions: The 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.

Keywords: Anorectal malformations; PCR; Reference gene; Stability; Variability.

Grants and funding

This study was supported by the National Natural Science Foundation of China (grant no. 81470788, 81770511), the Project of Key Laboratory of the Education Department of Liaoning (grant no. LS201601) and the Outstanding Scientific Fund of Shengjing Hospital (grant no. 201502). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.