Sds 7300 real time pcr

For the gene expression study, the extraction of total mRNA was performed from tissue testis using TRIZOL (Invitrogen, Milan, Italy) under sterile conditions, following the manufacturer's protocol. For each sample, 5 μg of mRNA was reverse-transcribed into cDNA through cDNA Archive High-Capacity Reverse Transcription kit (Life Technologies, Foster City, CA, USA). cDNA was stored at −20°C and it was used to quantify by TaqMan Gene Expression Assay (Life Technologies) the amount of NAIP (Rn01757809_m1; Life Technologies, Foster City, CA, USA) and survivin (Rn00574012_m1; Life Technologies, Foster City, CA, USA), using the instrument SDS 7300 Real-Time PCR (Applied Biosystems, Foster City, CA, USA). The result was expressed using 2^−ΔΔCT method (normalized versus varicocele + vehicle). β-actin (Rn00667869_m1; Life Technologies, Foster City, CA, USA) was used as endogenous control.

For the gene expression study, the extraction of total mRNA was performed from tissue prostate using TRIZOL (Invitrogen, Milan, Italy) under sterile conditions, following the manufacturer’s protocol. For each sample, 5 μg of mRNA were reverse transcripted into cDNA and 3 μl of cDNA were amplified in duplicate with the TaqMan Universal PCR Master Mix containing primer and specific probes (TaqMan), then using the instrument SDS 7300 Real Time PCR (Applied Biosystems, Foster City, CA). The reversed transcribed product was used to quantify the amount of IL-6. The result was expressed as number of copies of the target gene compared to the housekeeping gene (β-actin).