Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characte...

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Autores principales:	Juan Zhao, Cheng Wang, Lin Zhang, Aiai Lei, Linjie Wang, Lili Niu, Siyuan Zhan, Jiazhong Guo, Jiaxue Cao, Li Li, Hongping Zhang, Tao Zhong
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	caprine rumen genome-wide reference gene RT-qPCR Veterinary medicine SF600-1100 Zoology QL1-991
Acceso en línea:	https://doaj.org/article/f82465b31d1a4cbdb9c30dc921c26992
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spelling	oai:doaj.org-article:f82465b31d1a4cbdb9c30dc921c269922021-11-25T16:16:44ZGenome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen10.3390/ani111131372076-2615https://doaj.org/article/f82465b31d1a4cbdb9c30dc921c269922021-11-01T00:00:00Zhttps://www.mdpi.com/2076-2615/11/11/3137https://doaj.org/toc/2076-2615As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except <i>GAPT4</i>, indicating that they were stably expressed in the rumen tissues throughout development. <i>RPS4X</i> and <i>RPS6</i> were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (<i>IGF</i>1 and <i>TOP2A</i>), normalized by the selected most stable RGs (<i>RPS4X</i> and <i>RPS6</i>), were consistent with the results of RNA sequencing, while the use of <i>GAPDH</i> and <i>ACTB</i> as RGs resulted in altered profiles. Overall, <i>RPS4X</i> and <i>RPS6</i> showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.Juan ZhaoCheng WangLin ZhangAiai LeiLinjie WangLili NiuSiyuan ZhanJiazhong GuoJiaxue CaoLi LiHongping ZhangTao ZhongMDPI AGarticlecaprinerumengenome-widereference geneRT-qPCRVeterinary medicineSF600-1100ZoologyQL1-991ENAnimals, Vol 11, Iss 3137, p 3137 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	caprine rumen genome-wide reference gene RT-qPCR Veterinary medicine SF600-1100 Zoology QL1-991
spellingShingle	caprine rumen genome-wide reference gene RT-qPCR Veterinary medicine SF600-1100 Zoology QL1-991 Juan Zhao Cheng Wang Lin Zhang Aiai Lei Linjie Wang Lili Niu Siyuan Zhan Jiazhong Guo Jiaxue Cao Li Li Hongping Zhang Tao Zhong Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen
description	As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except <i>GAPT4</i>, indicating that they were stably expressed in the rumen tissues throughout development. <i>RPS4X</i> and <i>RPS6</i> were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (<i>IGF</i>1 and <i>TOP2A</i>), normalized by the selected most stable RGs (<i>RPS4X</i> and <i>RPS6</i>), were consistent with the results of RNA sequencing, while the use of <i>GAPDH</i> and <i>ACTB</i> as RGs resulted in altered profiles. Overall, <i>RPS4X</i> and <i>RPS6</i> showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.
format	article
author	Juan Zhao Cheng Wang Lin Zhang Aiai Lei Linjie Wang Lili Niu Siyuan Zhan Jiazhong Guo Jiaxue Cao Li Li Hongping Zhang Tao Zhong
author_facet	Juan Zhao Cheng Wang Lin Zhang Aiai Lei Linjie Wang Lili Niu Siyuan Zhan Jiazhong Guo Jiaxue Cao Li Li Hongping Zhang Tao Zhong
author_sort	Juan Zhao
title	Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen
title_short	Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen
title_full	Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen
title_fullStr	Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen
title_full_unstemmed	Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen
title_sort	genome-wide identification of reference genes for reverse-transcription quantitative pcr in goat rumen
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/f82465b31d1a4cbdb9c30dc921c26992
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Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

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