Abi 7900ht rt pcr system

Total RNA was extracted from the gonads of male GF and testis-like GFYT hybrids (n = 3 each) with TRIzol™ Reagent (Invitrogen, Carlsbad, CA, United States). The concentration and purity of RNA were determined using spectrophotometry and agarose gel electrophoresis. After treatment with RNase-free DNase (Promega, Madison, WI, United States), the total RNA was reverse transcribed to complementary DNA (cDNA) with Rever Tra Ace M-MLV (TOYOBO, Osaka, Japan). RT-qPCR was carried out by using 23SYBR Green mix (TOYOBO, Osaka, Japan) and ABI 7900HT RT-PCR System (Applied Biosystems, Carlsbad, CA, United States). β-actin was used as the internal reference and the expression levels were calculated with the 2^−ΔΔCt method. The primers used here were listed in Table 1.

After establishing RNA purity (OD ratio of 260/280), the samples were subjected to reverse transcription using MegaPlex RT Primers and TaqMan miRNA reverse transcription kits (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. The cDNA was diluted in Universal PCR master mix-II (Applied Biosystems) and then loaded on to TaqMan^® Low Density Array (TLDA) microfluidic MicroRNA 384-well cards (Applied Biosystems) for real-time PCR (ABI 7900 HT RT-PCR System). The relative concentration of miRNAs was calculated by comparative (RQ = 2^−ΔΔCt) analysis and the fold change determined as log10 RQ. The data were analyzed using RQ Manager 12.1 (Applied Biosystems) and RealTime StatMiner software (Integromics, Philadelphia, PA, USA).

Reverse transcription (RT) was performed using Superscript III (Invitrogen, Waltham, MA, USA) with an initial concentration of 5 μg total RNA. We established an in-house human panel for ROS profiling analysis using real-time qPCR with SYBR green reagents (Applied Biosystems, Foster City, CA, USA). The thermocycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min for 40 cycles, on the ABI 7900 HT RT-PCR system (Applied Biosystems). Each sample was analyzed in duplicate. Relative expression values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative gene expressions were calculated using the 2^−ΔΔCt method, ΔCt = Ct (target gene) − Ct (GAPDH), where Ct indicates the cycle threshold (the fractional cycle number at which the fluorescent signal reaches the detection threshold). The primer sequences for the selected 52 genes are listed in Table 4.