Determination of steroidogenesis-related genes using quantitative real-time PCR

The epididymides obtained from the rats were dissected and then frozen in liquid nitrogen. Total RNA was extracted after homogenization using the standard TRIzol® reagent extraction method (Invitrogen, USA). The RNA concentrations were determined at 260/280 nm using an ultraviolet spectrophotometer. Purified RNA obtained from a sample containing 500 ng of total RNA was immediately transcribed into single-stranded cDNA using a First Strand cDNA Synthesis Kit (MBI Fermentas, Germany) according to the manufacturer’s directions. Reverse transcription (RT) was performed at a total volume of 25 µl using 0.5 µl poly (dT)18 primer and 13 µl RNA.The reaction was run at 37 °C for 90 min and ended with a denaturation step at 70 °C for 15 min. The tubes containing the cDNA were stored at − 20 °C.

The RT-PCR analyses for StAR, 3β-HSD, and cholesterol side-chain cleavage enzyme CYP450scc (CYP11A1 gene; Leydig cell-specific biomarker) were performed on an RT-PCR detection system (iQ5-Bio-Rad Laboratories, Cepheid, USA) using Syber green PCR master mix (TaKaRa Biotech Co., Ltd.). The expression levels of the gene mRNAs were normalized to a β-actin housekeeping gene (Actb). Our target gene and the Actb oligonucleotide sequence (Table ​(Table1)1) were based from published literatures^24,25. The PCR reactions were performed in 25 μl reaction mixtures containing 12.5 μl 1 × SYBR, 0.5 μl forward primers, 0.5 μl reverse primer, 6.5 μl distilled water, and 5 μl cDNA template. The amplification cycle started with a preliminary denaturation step at 95 °C for 3 min followed by 35 cycles of denaturation at 95 °C for 15 s and then by an annealing step at 55 °C for 30 s. Finally, an extension step was performed at 72 °C for 30 s. Samples and controls were run in duplicate. The amplification was followed by a melt curve analysis to ensure that no primer–dimer amplification occurred. The gene expression levels were calculated using the formulae provided by Bio-Rad Laboratories, Inc.:Ef = 10 − 1/slope; Efficiency (%) = (Ef − 1) × 100²⁶. We performed a relative quantification of the target to the reference by using the ΔC_T method provided that the E for our target genes and the reference primer (ACTB) are the same, that is, Ratio_{(reference/target)} = Ef^{CT(reference)−CT(target)}.

Primer sequences for RT-PCR.

F: TCT CTA GTG TCT CCC ACT GCA TAG C

R: TTA GCA TCC CCT GTT CG TAG CT

F: ACAT GGC CAA GAT GGT ACA GTT G

R: ACG AAG CAC CAG GTC ATT CAC

F: ACAT GGC TCT GGG AGT TAT AAG GT

R: TTA GTG ACT GGC AAG GCT TCT G

AGA AGA TCT GGC ACC ACA CC

TAC GAC CAG AGG CAT ACA GG

Abbreviations: F: forward primer; R: reverse primer. StAR: steroidogenic acute regulatory protein; CYP11A1: P450scc-cholesterol side-chain cleavage enzyme; 3β-HSD: 3β-hydroxysteroid dehydrogenase-1.