Thermal cycler dice real time system software ver 5

Genomic DNA was eliminated from total RNA by TURBO DNA-free™ Kit (Thermo Fisher Scientific, Waltham, MA). First-strand cDNA was synthesized from 1 μg of total RNA using the SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific) with random hexamers. The PCR reaction mixture consisted of each primer pair (0.2 μM) and GoTaq® qPCR Master Mix (Promega, Madison, WI). Primer sequences and annealing temperature are shown in Supplemental Table S5. Data were analyzed using the Thermal Cycler Dice® TP800 (Takara, Shiga, Japan) and Thermal Cycler Dice® Real Time System Software Ver.5.11. Dissociation curve analysis was performed to ensure only a single peak was amplified. The expression levels of mRNA were normalized to EF1α in respective samples. Data were expressed Z-score and means ± SEM.

Testes from mice in each group (n = 5) were examined. The iCycler thermal cycler (Bio-Rad, Hercules, CA, USA) was used to perform the PCR reactions, and the mixtures were stored at −80 °C until analysis. Real-time RT-PCR was performed on 3 ng cDNA using the validated SYBR Green gene expression assay in combination with SYBR Premix Ex Taq^TM (TaKaRa, Bio Inc., Ohtsu, Japan) for measuring 13 germ cell genes (Stra8, Spo11, Tnp1,cKit, Gfra1, Oct4, PLZF, Sycp3, Ddx4, Boll, Crem, Prm1, and Acrosin), 28 Sertoli cell genes (Amh, Aqp8, Ccnd2, Clu,Claudin-11,Cst12, Cst9, Dhh, Espn, Fshr, Fyn, GATA1,Inhba,Inhbb, Msi1,Occlidin,Rhox5, Testin, Spata2,shbg,Sox9, sympk, Tjp1, Trf, Wt1,Wnt5a, ZO-1, and ZO-2) and GAPDH. Quantitative real-time PCR was performed in duplicate using the Thermal Cycler Dice Real-time System TP800 (TaKaRa). The Thermal Cycler Dice Real-time System software Ver.5.1.1 (TaKaRa) was used to analyze the data, and the comparative C_t method (2^-∆∆Ct) was used to quantify gene expression levels. The results are expressed relative to the amount of GAPDH transcript used as an internal control. Relative mRNA intensity was calculated, and the expression in the control group for each point was normalized to 1. Data are presented as the mean ± standard deviation (SD). The primers used in the analysis are listed in Table 1.