Identification of Suitable Reference Genes for Gene Expression Studies of Shoulder Instability

Identification of Suitable Reference Genes for Gene Expression Studies of Shoulder Instability

Author Leal, Mariana Ferreira Autor UNIFESP Google Scholar
Belangero, Paulo Santoro Autor UNIFESP Google Scholar
Cohen, Carina Autor UNIFESP Google Scholar
Figueiredo, Eduardo Antonio Autor UNIFESP Google Scholar
Loyola, Leonor Casilla Autor UNIFESP Google Scholar
Pochini, Alberto Castro Autor UNIFESP Google Scholar
Smith, Marilia Cardoso Autor UNIFESP Google Scholar
Andreoli, Carlos Vicente Autor UNIFESP Google Scholar
Belangero, Sintia Iole Autor UNIFESP Google Scholar
Ejnisman, Benno Autor UNIFESP Google Scholar
Cohen, Moises Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract Shoulder instability is a common shoulder injury, and patients present with plastic deformation of the glenohumeral capsule. Gene expression analysis may be a useful tool for increasing the general understanding of capsule deformation, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. Although RT-qPCR is highly sensitive and specific, it requires the use of suitable reference genes for data normalization to guarantee meaningful and reproducible results. in the present study, we evaluated the suitability of a set of reference genes using samples from the glenohumeral capsules of individuals with and without shoulder instability. We analyzed the expression of six commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, TBP and TFRC) in the antero-inferior, antero-superior and posterior portions of the glenohumeral capsules of cases and controls. the stability of the candidate reference gene expression was determined using four software packages: NormFinder, geNorm, BestKeeper and DataAssist. Overall, HPRT1 was the best single reference gene, and HPRT1 and B2M composed the best pair of reference genes from different analysis groups, including simultaneous analysis of all tissue samples. GenEx software was used to identify the optimal number of reference genes to be used for normalization and demonstrated that the accumulated standard deviation resulting from the use of 2 reference genes was similar to that resulting from the use of 3 or more reference genes. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1. Although the use of different reference gene combinations yielded variable normalized quantities, the relative quantities within sample groups were similar and confirmed that no obvious differences were observed when using 2, 3 or 4 reference genes. Consequently, the use of 2 stable reference genes for normalization, especially HPRT1 and B2M, is a reliable method for evaluating gene expression by RT-qPCR.
Language English
Sponsor Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Date 2014-08-14
Published in Plos One. San Francisco: Public Library Science, v. 9, n. 8, 9 p., 2014.
ISSN 1932-6203 (Sherpa/Romeo, impact factor)
Publisher Public Library Science
Extent 9
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000341017000084

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