Identification of suitable reference genes for gene expression studies of shoulder instability.

Mariana Ferreira Leal,Sintia Iole Belangero,Leonor Casilla Loyola,Marília Cardoso Smith,Carlos Vicente Andreoli,Moises Cohen,Eduardo Antônio Figueiredo,Alberto Castro Pochini,Benno Ejnisman,Paulo Santoro Belangero,Carina Cohen,Tim Douglas Aumann

doi:10.1371/journal.pone.0105002

Mariana Ferreira Leal, Sintia Iole Belangero + Show 10 more

Open Access

https://doi.org/10.1371/journal.pone.0105002

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Journal: PloS one	Publication Date: Aug 14, 2014
Citations: 45	License type: CC BY 4.0

Affiliation: Universidade Federal de São Paulo

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.

Highlights

Shoulder dislocation occurs in 1 to 2% of the population [1], and traumatic injuries account for 95% of shoulder dislocation episodes [2]
These shoulder injuries are frequently observed in young athletes that are involved in competitive sports [3], and shoulder instability (SI) is often observed after the initial episode of shoulder dislocation, with a recurrence rate of up to 100% in young athletes [4,5]
We hypothesized that misregulated expression of several genes may have a role in the capsular deformation observed in SI patients and that such molecular alterations may explain the high rate of shoulder dislocation recurrence after the first episode of traumatic dislocation

Summary

Introduction

Shoulder dislocation occurs in 1 to 2% of the population [1], and traumatic injuries account for 95% of shoulder dislocation episodes [2] These shoulder injuries are frequently observed in young athletes that are involved in competitive sports [3], and shoulder instability (SI) is often observed after the initial episode of shoulder dislocation, with a recurrence rate of up to 100% in young athletes [4,5]. After episodes of shoulder dislocation, SI patients present plastic deformation of the glenohumeral capsule [6,7]. The antero-inferior (AI) region of the capsule is the most frequently injured site [7,8], previous macroscopic analysis of the collagen fiber bundle architecture in the AI region of the glenohumeral capsule revealed that a system of bundles spirally crossing one another permits the entire capsule to resist tensile and shear loads [9]. Our group recently began investigating alterations in gene expression in SI, as gene expression analysis has previously been used to increase understanding of the molecular events involved in other traumatic sport injuries such as ligament [10,11] and tendon injuries (for a review, see [12])

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