Abstract
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 (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) 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. ACTB was the best single reference gene and ACTB+TBP 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 FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S 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 ACTB+TBP+18S+HPRT1 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.
Highlights
The anterior cruciate ligament (ACL) is an important structure in the knee and is one of the most frequently injured structures during high-impact sporting activities [1,2,3]
The results of the present study indicate that the use of suitable reference genes for reliable gene expression evaluation by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) should consider the type of ACL samples investigated
Based on the evaluation of different analysis groups, ACTB seems to be the most suitable reference gene and ACTB + TBP seems to be the best pair of reference genes
Summary
The anterior cruciate ligament (ACL) is an important structure in the knee and is one of the most frequently injured structures during high-impact sporting activities [1,2,3]. The ACL does not heal following lesions, and surgical reconstruction is the treatment of choice in most cases [4,5]. Reconstructive surgery aims to restore the kinematics and stability of the injured knee, which allows a return to sports and may help to prevent osteoarthritis in the long term [3,5,6,7]. An improved understanding of the regulation of gene expression in normal and injured ACL will be important for guiding patient management and the development of new therapeutic options complementary to surgery. Sensitivity, and capacity for high-throughput analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently considered to be the gold standard technique for evaluation of gene expression [13]; this technique is commonly used to validate data obtained by other methods [14]
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