Primer design software

QPCR was performed using a LightCycler 480 with a SYBR Green PCR Master Mix kit (Roche Diagnostics) as previously described (6 (link), 22 (link)). The primer sets used were designed using the Roche primer design software (Roche Diagnostics) and are listed in Table 1. QPCR cycling conditions: initial step at 95°C followed by 45 cycles at 95°C for 10 sec, 61°C for 10 sec, and 72°C for 10 sec. The comparative threshold cycle (C_t) method was used to analyze the data and 18S rRNA was used for data normalization. We initially determined the Ct values of three potential housekeeping genes, GAPDH, Tubulin, and 18S rRNA in cDNA samples from isolated gonocytes cultured for 1 day after siRNA interference, and 18S rRNA showed that it presented minimal changes in Ct values between samples. Assays were performed in triplicate. All experiments were performed using a minimum of three independent sample preparations and the mean ± SEM are plotted.

QPCR was performed using a LightCycler 480 with a SYBR Green PCR Master Mix kit (Roche Diagnostics). The primer sets used were designed using the Roche primer design software (Roche Diagnostics) and are listed in Table 3. QPCR cycling conditions were as follows: Initial step at 95 °C followed by 45 cycles at 95 °C for 10 s, 61 °C for 10 s, and 72 °C for 10 s. The direct detection of PCR products was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green dye to double-stranded DNA. The comparative threshold cycle (C_t) method was used to analyze the data and 18S rRNA was used for data normalization, as it was previously shown to be an adequate housekeeping gene for both rat germ cells and human seminomas [20 (link),44 (link)]. Assays were performed in triplicate. All experiments were performed using a minimum of three independent sample preparations, with each condition performed in duplicate, and the mean ± SEM are shown.