title: Identification of suitable reference genes for gene expression studies in tendons from patients with rotator cuff tear.
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.
authors.affiliation: 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.
authors.forename: Mariana Ferreira
authors.initials: MF
authors.lastname: Leal
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Paulo Santoro
authors.initials: PS
authors.lastname: Belangero
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Eduardo Antônio
authors.initials: EA
authors.lastname: Figueiredo
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Carina
authors.initials: C
authors.lastname: Cohen
authors.affiliation: 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.
authors.forename: Leonor Casilla
authors.initials: LC
authors.lastname: Loyola
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Carlos Vicente
authors.initials: CV
authors.lastname: Andreoli
authors.affiliation: Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Marília Cardoso
authors.initials: MC
authors.lastname: Smith
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Alberto
authors.initials: A
authors.lastname: de Castro Pochini
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Benno
authors.initials: B
authors.lastname: Ejnisman
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Moises
authors.initials: M
authors.lastname: Cohen
mesh_terms: D000328:Adult; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012015:Reference Standards; D000070636:Rotator Cuff Injuries; D000070599:Shoulder Injuries; D013708:Tendon Injuries / Q000235:genetics*