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.
Highlights
Rotator cuff degeneration is a very common orthopedic condition, and there are multiple factors that eventually lead to a full-thickness rotator cuff tear [1]
18S and B2M presented high standard deviation (SD) of Crt in the analysis involving all samples according to BestKeeper software (SD = 1.17, SD = 1.03, respectively), in which any studied gene with the SD higher than 1 can be considered inconsistent
When we individually evaluated each tendon region of patients with rotator cuff tears, we observed that HPRT1 was the most stable gene for the central cuff (CC) and posterior superior cuff (PC) samples
Summary
Rotator cuff degeneration is a very common orthopedic condition, and there are multiple factors that eventually lead to a full-thickness rotator cuff tear [1]. The incidence rate of degenerative rotator cuff tears increases with age; degenerative rotator cuff tears will become an increasingly prevalent clinical problem [2]. Surgical repair of tendon tears significantly improves pain and function; recurrent tearing of the rotator cuff is not infrequent [2]. Several studies have been performed to elucidate the molecular alterations involved in tendon tear and the failure of cuff healing (for a review, see [1,2,3,4]). An improved understanding of the regulation of gene expression in normal and injured tendons will be important for guiding patient management and the development of new therapeutic options complementary to surgery. As a result of its accuracy, sensitivity and capacity for high throughput analysis, reversetranscription quantitative polymerase chain reaction (RT-qPCR) is currently considered to be the gold standard technique for evaluation of gene expression [5]; this technique is commonly used to validate data obtained by other methods [6]
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