Identification of suitable reference genes for gene expression studies in tendons from patients with rotator cuff tear

Mariana Ferreira Leal; Paulo Santoro Belangero; Eduardo Antônio Figueiredo; Carina Cohen; Leonor Casilla Loyola; Carlos Vicente Andreoli; Marília Cardoso Smith; Alberto de Castro Pochini; Benno Ejnisman; Moises Cohen

doi:10.1371/journal.pone.0118821

Identification of suitable reference genes for gene expression studies in tendons from patients with rotator cuff tear

PLoS One. 2015 Mar 13;10(3):e0118821. doi: 10.1371/journal.pone.0118821. eCollection 2015.

Affiliations

¹ Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil; Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
² Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
³ Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

Abstract

Rotator cuff tear is one of the most common causes of shoulder dysfunction. Gene expression analysis may be a useful tool for understanding tendon tears and the failure of cuff healing, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluate the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using samples from the rotator cuff tendons of 28 individuals with tendon tears (3 tendons regions) and 8 controls (2 tendon regions); for the tear patients, we evaluated ruptured and non-ruptured tendon samples. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper and DataAssist software packages. Overall, HPRT1 was the best single reference gene, and HPRT1+TBP composed the best pair and HPRT1+TBP+ACTB composed the best trio of reference genes from the analysis of different groups, including the simultaneous analysis of all tissue samples. To identify the optimal combination of reference genes, we evaluated the expression of COL1A1 and COL3A1, and no obvious differences were observed when using 2, 3 or 4 reference genes for most of the analyses. However, COL3A1 expression differed between ruptured and non-ruptured (posterior superior region) tendons of patients only when normalized by HPRT1+TBP+B2M and HPRT1+TBP. On the other hand, the comparison between these two groups using the best trio of reference genes (HPRT1+TBP+ACTB) and 4 reference genes did not revealed a significant difference in COL3A1 expression. Consequently, the use of suitable reference genes for a reliable gene expression evaluation by RT-qPCR should consider the type of tendon samples investigated. HPRT1+TBP+ACTB seems to be the best combination of reference genes for the analysis of involving different tendon samples of individuals with rotator cuff tears.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adult
Female
Gene Expression Profiling / methods*
Gene Expression Profiling / standards
Humans
Male
Real-Time Polymerase Chain Reaction / methods
Real-Time Polymerase Chain Reaction / standards
Reference Standards
Rotator Cuff Injuries*
Shoulder Injuries*
Tendon Injuries / genetics*

Grants and funding

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; MC and MCS) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; LCL, MC and MFL) as grants and fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.