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Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485207/ https://www.ncbi.nlm.nih.gov/pubmed/31065464 http://dx.doi.org/10.7717/peerj.6829 |
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author | Long, Caiyun Xiao, Yunxia Li, Siying Tang, Xiaobing Yuan, Zhengwei Bai, Yuzuo |
author_facet | Long, Caiyun Xiao, Yunxia Li, Siying Tang, Xiaobing Yuan, Zhengwei Bai, Yuzuo |
author_sort | Long, Caiyun |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6485207 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-64852072019-05-07 Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations Long, Caiyun Xiao, Yunxia Li, Siying Tang, Xiaobing Yuan, Zhengwei Bai, Yuzuo PeerJ Developmental Biology 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. PeerJ Inc. 2019-04-23 /pmc/articles/PMC6485207/ /pubmed/31065464 http://dx.doi.org/10.7717/peerj.6829 Text en © 2019 Long et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Developmental Biology Long, Caiyun Xiao, Yunxia Li, Siying Tang, Xiaobing Yuan, Zhengwei Bai, Yuzuo Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations |
title | Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations |
title_full | Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations |
title_fullStr | Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations |
title_full_unstemmed | Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations |
title_short | Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations |
title_sort | identification of optimal endogenous reference rnas for rt-qpcr normalization in hindgut of rat models with anorectal malformations |
topic | Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485207/ https://www.ncbi.nlm.nih.gov/pubmed/31065464 http://dx.doi.org/10.7717/peerj.6829 |
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