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

Best reference gene for each group of sample.

<p>^aBest reference gene determined considering the intragroup and intergroup variation.</p><p>*When the intragroup and intergroup variation was not considered, <i>ACTB</i> was the best reference gene by NormFinder. Bold letters: best pairs of reference genes by more than one of the methods commonly used (Normfinder, GeNorm, BestKeeper, DataAssist, ΔCt method and RefFinder). ACL: anterior cruciate ligament.</p><p>Best reference gene for each group of sample.</p

<i>FN1</i> (A) and <i>PLOD1</i> (B) expression normalized by different combinations of candidate reference genes in anterior cruciate ligament specimens.

<p><i>18S</i>: target expression normalized by <i>18S</i>; <i>ACTB</i>: target expression normalized by <i>ACTB</i>; <i>GAPDH</i>: target expression normalized by <i>GAPDH; A_T</i>: target expression normalized by <i>ACTB + TBP</i>; <i>A_18S</i>: target expression normalized by <i>ACTB + 18S</i>; <i>A_T_18S</i>: <i>FN1</i> expression normalized by <i>ACTB + TBP + 18S</i>; <i>A_H_18S</i>: target expression normalized by <i>ACTB + HPRT1 + 18S</i>; <i>A_T_H_18S</i>: target expression normalized by <i>ACTB + TBP + HPRT1 + 18S</i>; ACL-I: isolated anterior cruciate ligament tear samples; ACL-M: anterior cruciate ligament tear samples of patients with concomitant meniscal tear; ACL-C: anterior cruciate ligament samples of controls.</p

Distribution of the clinical outcomes of anterior cruciate ligament tear patients and controls.

<p>N: number of samples; SD: standard deviation.</p><p>Distribution of the clinical outcomes of anterior cruciate ligament tear patients and controls.</p

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

<p>Lower values of accumulated standard deviation (Acc.SD) indicate the optimal number of reference gene as estimated by the GenEx software package. ACL-I: isolated anterior cruciate ligament tear samples; ACL-M: anterior cruciate ligament tear samples of patients with concomitant meniscal tear; ACL-C: anterior cruciate ligament samples of controls.</p