Evaluation of the Most Stable Reference Genes in Testicular Tissue of the Men with Azoospermia

Real-time quantitative reverse transcriptase- polymerase chain reaction (qRT-PCR), is a fast, sensitive and reliable method of gene expression comparison that is prone to a lot of technical errors. On the other hand, historical reference (housekeeping) genes are not suitable for all tissues. Herein, we have tried to identify and evaluate the best reference gene for testis tissues for further qRT-PCR experiments.
Testis tissues of 15 men with non-obstructive (NOA) and 15 men with obstructive (OA) azoospermia (as control individuals) were collected. Primer designing and verification of four candidate reference genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L37 (RPL37), ring finger protein 1 (RING1) and eukaryotic translation elongation factor 2 (eEF2) were performed using Beacon designer 8.1 software. PCR pre-optimization for reverse transcriptase input RNA and best primer concentration were included. Melt curve analysis was drawn and values of quantitation cycle (Cq) were extracted. Mean Cq analysis was calculated using BestKeeper v1 software and suitable reference genes were selected afterward.
Findings: Comparing the mean Cq values between the NOA and OA groups declared that RPL37 and GAPDH showed the lowest standard deviations of 1.39 and 1.67 among the other candidates. GAPDH and RPL37 were selected as the best reference genes in testis tissues with their r values of 0.959 and 0.927, respectively.
The results of this study show that the best reference genes for normalization of qRT-PCR data of testis tissues are GAPDH and RPL37.