title: Identification of suitable reference genes for gene expression studies of shoulder instability.
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.
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil; Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, 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, São Paulo, 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, São Paulo, Brazil.
authors.forename: Carina
authors.initials: C
authors.lastname: Cohen
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, 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, São Paulo, Brazil; Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, 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, São Paulo, Brazil.
authors.forename: Alberto Castro
authors.initials: AC
authors.lastname: Pochini
authors.affiliation: Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, 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, São Paulo, Brazil.
authors.forename: Carlos Vicente
authors.initials: CV
authors.lastname: Andreoli
authors.affiliation: Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Ferderal de São Paulo, São Paulo, São Paulo, Brazil; Laboratório Interdisciplinar de Neurociência Clínica, Departamento de Psiquiatria, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Sintia Iole
authors.initials: SI
authors.lastname: Belangero
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Benno
authors.initials: B
authors.lastname: Ejnisman
authors.affiliation: Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Moises
authors.initials: M
authors.lastname: Cohen
mesh_terms: D000293:Adolescent; D000328:Adult; D005260:Female; D015870:Gene Expression; D006801:Humans; D008297:Male; D060888:Real-Time Polymerase Chain Reaction; D012783:Shoulder Dislocation / Q000235:genetics*; D055815:Young Adult