Comprehensive Selection of Reference Genes for Expression Studies in Meniscus Injury Using Quantitative Real-time PCR Mariana Ferreira Leal 1, 2, * , Gustavo Gonçalves Arliani 1 , Diego Costa Astur 1 , Carlos Eduardo Franciozi 1 , Pedro Debieux 1 , Carlos Vicente Andreoli 1 , Marília Cardoso Smith 2 , Alberto de Castro Pochini 1 , Benno Ejnisman 1 , Moises Cohen 1 1 Departamento de Ortopedia e Traumatologia, Universidade Federal de São Paulo, 04038-032, São Paulo, SP, Brazil 2 Disciplina de Genética, Departamento de Morfologia e Genética, Universidade Federal de São Paulo, 04023-001, São Paulo, SP, Brazil * Corresponding Author. E-mail: satarupa@smst.iitkgp.ernet.in Abstract. The meniscus plays critical roles in the knee function. Meniscal tears can lead to knee osteoarthritis. Gene expression analysis may be a useful tool for understanding meniscus tears, 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. We evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1 and TBP) using meniscus samples of (1) 19 patients with isolated meniscal tears, (2) 20 patients with meniscal tears and combined anterior cruciate ligament injury (ACL), and (3) 11 controls without meniscal tears. The stability of the candidate reference genes was determined using the NormFinder, geNorm, BestKeeper DataAssist and RefFinder software packages and comparative ΔCt method. Overall, HPRT1 was the best single reference gene. However, GenEx software demonstrated that two or more reference genes should be used for gene expression normalization, which was confirmed when we evaluated TGFβR1 expression using several reference gene combinations. HPRT1 + TBP was the most frequently identified pair from the analysis of samples of (1) meniscal tear samples of patients with a concomitant ACL tears, (2) all meniscal tears, and (3) all samples. HPRT1 + GAPDH was the most frequently identified pair from the analysis of samples of isolated meniscal tear samples and controls. In the analysis involving only controls, GAPDH + 18S was the most frequently identified pair. In the analysis of only isolated meniscal tear samples and in the analysis of meniscal tear samples of patients with concomitant ACL tears and controls, both HPRT1 + TBP and HPRT1 + GAPDH were identified as suitable pairs. If the gene expression study aims to compare non-injured meniscus, isolated meniscal tears and meniscal tears of patients with ACL tears as three independent groups, the trio of HPRT1 + TBP + GAPDH is the most suitable combination of reference genes. Keywords: ACL, anterior cruciate ligament; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MRI, magnetic resonance imaging; TLDA, TaqMan Low-Density Array; AAV, adeno-associated virus; Crt, relative cycle threshold; Ct, cycle threshold; SD, standard deviation; CV, coefficient of variance; RQ, relative quantification; Acc.SD, accumulated standard deviation. Keywords: Knee injury; Meniscal tears; Gene expression; Reverse-transcription quantitative polymerase chain reaction; Expression normalization; Reference genes. 1. Introduction Menisci are important components in joint biomechanics with crucial roles in the knee joint: distributing joint forces, load bearing, and enhancing joint stability (Lee et al., 2014 and Kaleka et al., 2014). Lesion of this structure can cause pain, joint swelling, and osteoarthritis in the long term. Younger people are more likely to have acute lesions due to trauma, whereas older people are more likely to have lesions due to degeneration (Englund et al., 2009, Pauli et al., 2011 and Rai et al., 2013). Patients with traumatic meniscal tears commonly present an associated rupture of the anterior Mariana Ferreira Leal, Gustavo Gonçalves Arliani, Diego Costa Astur, et al. Biomed. Lab. Clin. Res., 2019, 4(4): 36-44. 36