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>