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

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.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol & Genet, Disciplina Genet, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psiquiatria, Lab Interdisciplinar Neurociencia Clin, São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morfol & Genet, Disciplina Genet, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psiquiatria, Lab Interdisciplinar Neurociencia Clin, São Paulo, BrazilWeb of Scienc

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<p>^ap- value by paired T-test</p><p>^bp-value by independent T-test</p><p>*p < 0.05. SD: standard deviation; CC: central cuff (injured supraspinatus tendon) samples of cases; PC: posterior superior cuff samples of cases; AC: anterior cuff samples of cases; CCC: central cuff samples of controls; ACC: anterior cuff samples of controls.</p><p><i>COL3A1</i> expression normalized by different combinations of reference genes in the rotator cuff tendons samples.</p

Identification of Suitable Reference Genes for Gene Expression Studies in Tendons from Patients with Rotator Cuff Tear

<div><p>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 (<i>18S</i>, <i>ACTB</i>, <i>B2M</i>, <i>GAPDH</i>, <i>HPRT1</i> and <i>TBP</i>) 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, <i>HPRT1</i> was the best single reference gene, and <i>HPRT1+TBP</i> composed the best pair and <i>HPRT1+TBP+ACTB</i> 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 <i>COL1A1</i> and <i>COL3A1</i>, and no obvious differences were observed when using 2, 3 or 4 reference genes for most of the analyses. However, <i>COL3A1</i> expression differed between ruptured and non-ruptured (posterior superior region) tendons of patients only when normalized by <i>HPRT1+TBP+B2M</i> and <i>HPRT1+TBP</i>. On the other hand, the comparison between these two groups using the best trio of reference genes (<i>HPRT1+TBP+ACTB</i>) and 4 reference genes did not revealed a significant difference in <i>COL3A1</i> 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. <i>HPRT1+TBP+ACTB</i> seems to be the best combination of reference genes for the analysis of involving different tendon samples of individuals with rotator cuff tears.</p></div

Best combination of reference genes for each group of sample.

<p>^aBest trio combination is based in a visual inspection of all the ranks generated by the four software.</p><p>*Best combination of two genes determined considering the intragroup and intergroup variation. CC: central cuff (injured supraspinatus tendon) samples of cases; PC: posterior superior cuff samples of cases; AC: anterior cuff samples of cases; CCC: central cuff samples of controls; ACC: anterior cuff samples of controls; Bold letters: best pairs of reference genes by more than one software.</p><p>Best combination of reference genes for each group of sample.</p

<i>COL1A1</i> (A) and <i>COL3A1</i> (B) expression normalized by different combinations of candidate reference genes in tendons specimens.

<p>HTAB: collagen expression normalized by <i>HPRT1 + TBP + ACTB + B2M</i>; HTA: collagen expression normalized by <i>HPRT1 + TBP + ACTB</i>; HTB: collagen expression normalized by <i>HPRT1 + TBP + B2M</i>; HT: collagen expression normalized by <i>HPRT1 + TBP</i>; HA: collagen expression normalized by <i>HPRT1 +</i> ACTB; HB: collagen expression normalized by <i>HPRT1 + B2M</i>; AB: collagen expression normalized by <i>ACTB + B2M</i>; A: collagen expression normalized by <i>ACTB</i>; G: collagen expression normalized by <i>GAPDH</i>; CC: central cuff (injured supraspinatus tendon) samples of cases; PC: posterior superior cuff samples of cases; AC: anterior cuff samples of cases; CCC: central cuff samples of controls; ACC: anterior cuff samples of controls.</p

Distribution of the clinical outcomes of rotator cuff tear patients and controls.

<p>^aTear size according to Cofield et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118821#pone.0118821.ref020" target="_blank">20</a>]. N: number of samples; SD: standard deviation.</p><p>Distribution of the clinical outcomes of rotator cuff tear patients and controls.</p

Accumulated standard deviation for the 6 reference genes in tendon samples.

<p>Lower values of accumulated standard deviation (Acc.SD) indicate the optimal number of reference gene as estimated by the GenEx software package. CC: central cuff (injured supraspinatus tendon) samples of cases; PC: posterior superior cuff samples of cases; AC: anterior cuff samples of cases; CCC: central cuff samples of controls; ACC: anterior cuff samples of controls.</p

Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR

<div><p>The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (<i>18S</i>, <i>ACTB</i>, <i>B2M</i>, <i>GAPDH</i>, <i>HPRT1</i>, and <i>TBP</i>) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. <i>ACTB</i> was the best single reference gene and <i>ACTB+TBP</i> was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of <i>FN1</i> and <i>PLOD1</i>. We observed that at least 3 reference genes should be used. <i>ACTB+HPRT1+18S</i> is the best trio for the analyses involving isolated ACL tears and controls. Conversely, <i>ACTB+TBP+18S</i> is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups <i>ACTB+TBP+18S+HPRT1</i> should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.</p></div

<i>COL1A1</i> expression normalized by different combinations of candidate reference genes in glenohumeral capsule specimens.

<p>AI: antero-inferior region of the glenohumeral capsule; AS: antero-superior region of the glenohumeral capsule; P: posterior region of the glenohumeral capsule.</p