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Endogenous reference genes for gene expression studies on bicuspid aortic valve associated aortopathy in humans

Endogenous reference genes for gene expression studies on bicuspid aortic valve associated aortopathy in humans
Endogenous reference genes for gene expression studies on bicuspid aortic valve associated aortopathy in humans
Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant regardless of aortic valve morphology, aortic diameter and other factors associated with aortopathy. Here, we identified an appropriate set of HK genes to be used as endogenous reference for quantifying gene expression in ascending aortic tissue using a spin column-based RNA extraction method. Ascending aortic biopsies were collected intra-operatively from patients undergoing aortic valve and/or ascending aortic surgery. These patients had BAV or tricuspid aortic valve (TAV), and the aortas were either dilated (?4.5cm) or undilated. The cohort had an even distribution of gender, valve disease and hypertension. The expression stability of 12 reference genes were investigated (ATP5B, ACTB, B2M, CYC1, EIF4A2, GAPDH, SDHA, RPL13A, TOP1, UBC, YWHAZ, and 18S) using geNorm software. The most stable HK genes were found to be GAPDH, UBC and ACTB. Both GAPDH and UBC demonstrated relative stability regardless of valve morphology, aortic diameter, gender and age. The expression of B2M and SDHA were found to be the least stable HK genes. We propose the use of GAPDH, UBC and ACTB as reference genes for gene expression studies of BAV aortopathy using ascending aortic tissue.
1932-6203
1-9
Harrison, Oliver
1feda5d5-8833-4587-9800-93135b68135a
Moorjani, Narain
ce44e232-2a6e-4616-8341-837ad0446acb
Torrens, Christopher
15a35713-0651-4249-8227-5901e2cfcd22
Ohri, Sunil
8aa5698c-78cf-4f59-a5af-5afa46f0348c
Cagampang, Felino
7cf57d52-4a65-4554-8306-ed65226bc50e
Harrison, Oliver
1feda5d5-8833-4587-9800-93135b68135a
Moorjani, Narain
ce44e232-2a6e-4616-8341-837ad0446acb
Torrens, Christopher
15a35713-0651-4249-8227-5901e2cfcd22
Ohri, Sunil
8aa5698c-78cf-4f59-a5af-5afa46f0348c
Cagampang, Felino
7cf57d52-4a65-4554-8306-ed65226bc50e

Harrison, Oliver, Moorjani, Narain, Torrens, Christopher, Ohri, Sunil and Cagampang, Felino (2016) Endogenous reference genes for gene expression studies on bicuspid aortic valve associated aortopathy in humans. PLoS ONE, 11 (10), 1-9. (doi:10.1371/journal.pone.0164329). (PMID:27727313)

Record type: Article

Abstract

Bicuspid aortic valve (BAV) disease is the most common congenital cardiac abnormality and predisposes patients to life-threatening aortic complications including aortic aneurysm. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most commonly used methods to investigate underlying molecular mechanisms involved in aortopathy. The accuracy of the gene expression data is dependent on normalization by appropriate housekeeping (HK) genes, whose expression should remain constant regardless of aortic valve morphology, aortic diameter and other factors associated with aortopathy. Here, we identified an appropriate set of HK genes to be used as endogenous reference for quantifying gene expression in ascending aortic tissue using a spin column-based RNA extraction method. Ascending aortic biopsies were collected intra-operatively from patients undergoing aortic valve and/or ascending aortic surgery. These patients had BAV or tricuspid aortic valve (TAV), and the aortas were either dilated (?4.5cm) or undilated. The cohort had an even distribution of gender, valve disease and hypertension. The expression stability of 12 reference genes were investigated (ATP5B, ACTB, B2M, CYC1, EIF4A2, GAPDH, SDHA, RPL13A, TOP1, UBC, YWHAZ, and 18S) using geNorm software. The most stable HK genes were found to be GAPDH, UBC and ACTB. Both GAPDH and UBC demonstrated relative stability regardless of valve morphology, aortic diameter, gender and age. The expression of B2M and SDHA were found to be the least stable HK genes. We propose the use of GAPDH, UBC and ACTB as reference genes for gene expression studies of BAV aortopathy using ascending aortic tissue.

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Accepted/In Press date: 25 September 2016
e-pub ahead of print date: 11 October 2016
Published date: 11 October 2016
Organisations: Human Development & Health

Identifiers

Local EPrints ID: 402540
URI: http://eprints.soton.ac.uk/id/eprint/402540
ISSN: 1932-6203
PURE UUID: e259ff63-8e29-4a0f-9852-8bf1f7ee4b90
ORCID for Felino Cagampang: ORCID iD orcid.org/0000-0003-4404-9853

Catalogue record

Date deposited: 10 Nov 2016 14:26
Last modified: 07 Oct 2020 01:47

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