Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models

Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of fre...

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Autores principales:	Juan J. Muñoz, Ana C. Anauate, Andressa G. Amaral, Frederico M. Ferreira, Elieser H. Watanabe, Renata Meca, Milene S. Ormanji, Mirian A. Boim, Luiz F. Onuchic, Ita P. Heilberg
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a5acc516851e4171b13e8bd2777a6a00
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spelling	oai:doaj.org-article:a5acc516851e4171b13e8bd2777a6a002021-12-02T17:13:17ZPpia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models10.1038/s41598-021-99366-x2045-2322https://doaj.org/article/a5acc516851e4171b13e8bd2777a6a002021-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-99366-xhttps://doaj.org/toc/2045-2322Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used “housekeeping” genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10–12 weeks old (Pkd1 flox/flox:Nestin cre/Pkd1 flox/−:Nestin cre, n = 10) and non-cystic (NC) controls (Pkd1 flox/flox/Pkd1 flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1 +/−, n = 6) and wild-type (WT) controls (Pkd1 +/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1 V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.Juan J. MuñozAna C. AnauateAndressa G. AmaralFrederico M. FerreiraElieser H. WatanabeRenata MecaMilene S. OrmanjiMirian A. BoimLuiz F. OnuchicIta P. HeilbergNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Juan J. Muñoz Ana C. Anauate Andressa G. Amaral Frederico M. Ferreira Elieser H. Watanabe Renata Meca Milene S. Ormanji Mirian A. Boim Luiz F. Onuchic Ita P. Heilberg Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models
description	Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used “housekeeping” genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10–12 weeks old (Pkd1 flox/flox:Nestin cre/Pkd1 flox/−:Nestin cre, n = 10) and non-cystic (NC) controls (Pkd1 flox/flox/Pkd1 flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1 +/−, n = 6) and wild-type (WT) controls (Pkd1 +/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1 V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.
format	article
author	Juan J. Muñoz Ana C. Anauate Andressa G. Amaral Frederico M. Ferreira Elieser H. Watanabe Renata Meca Milene S. Ormanji Mirian A. Boim Luiz F. Onuchic Ita P. Heilberg
author_facet	Juan J. Muñoz Ana C. Anauate Andressa G. Amaral Frederico M. Ferreira Elieser H. Watanabe Renata Meca Milene S. Ormanji Mirian A. Boim Luiz F. Onuchic Ita P. Heilberg
author_sort	Juan J. Muñoz
title	Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models
title_short	Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models
title_full	Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models
title_fullStr	Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models
title_full_unstemmed	Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models
title_sort	ppia is the most stable housekeeping gene for qrt-pcr normalization in kidneys of three pkd1-deficient mouse models
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/a5acc516851e4171b13e8bd2777a6a00
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Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models

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