Primer premier 7

Each sperm pellet was homogenized in 500 μL of TRIzol reagent at room temperature for 30 minutes. Then 200 μL of chloroform was added and shaken for 15 seconds, following centrifugation at 12,000 × g for 15 minutes at 4 °C. The upper supernatant was collected and precipitated using 500 μL isopropanol. After centrifugation of 12,000 × g, pellet was washed by 70% ethyl alcohol and resolved in sterile diethypyrocarbonate (DEPC) water. Complementary DNAs (cDNAs) were synthesized according to the instructions provided using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). Primers were designed using Primer Premier 7.0 software (PREMIER Biosoft International, Palo Alto, CA) and each primer was submitted into an National Center for Biotechnology Information (NCBI) BLAST search to ensure specificity for the target mRNA. Real-time RT-PCR was performed under conditions of 2 minutes at 95 °C, followed by 15 seconds at 95 °C and 50 seconds at 65 °C for 40 cycles. Data were analyzed using the GeneAmp5700 Sequence Detection System software (version 1.1; Applied Biosystems, Foster City, CA) and were converted into threshold cycle (CT) values. All samples were normalized according to b-actin content. The formula 2^–△Ct was used to calculate the relative mRNA levels.^{[20 (link)]}