Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.

Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 pot...

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Autores principales:	Zhichen Zhai, Yongchang Yao, Yingjun Wang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/74fc80a2aede410dbd4d481ab44b791a
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spelling	oai:doaj.org-article:74fc80a2aede410dbd4d481ab44b791a2021-11-18T07:45:03ZImportance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.1932-620310.1371/journal.pone.0064786https://doaj.org/article/74fc80a2aede410dbd4d481ab44b791a2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23705012/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.Zhichen ZhaiYongchang YaoYingjun WangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 5, p e64786 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Zhichen Zhai Yongchang Yao Yingjun Wang Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.
description	Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.
format	article
author	Zhichen Zhai Yongchang Yao Yingjun Wang
author_facet	Zhichen Zhai Yongchang Yao Yingjun Wang
author_sort	Zhichen Zhai
title	Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.
title_short	Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.
title_full	Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.
title_fullStr	Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.
title_full_unstemmed	Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.
title_sort	importance of suitable reference gene selection for quantitative rt-pcr during atdc5 cells chondrocyte differentiation.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/74fc80a2aede410dbd4d481ab44b791a
work_keys_str_mv	AT zhichenzhai importanceofsuitablereferencegeneselectionforquantitativertpcrduringatdc5cellschondrocytedifferentiation AT yongchangyao importanceofsuitablereferencegeneselectionforquantitativertpcrduringatdc5cellschondrocytedifferentiation AT yingjunwang importanceofsuitablereferencegeneselectionforquantitativertpcrduringatdc5cellschondrocytedifferentiation
_version_	1718423043361996800

Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.

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