2.5 Isolation of RNA，cDNA Synthesis and Real-Time ...
Total RNA was isolated using TRIzol reagent(CoWin Bioscience，Beijing，China).The quality of the isolated RNA was assessed by electrophoresis on 1% agarose gels based on the integrity of 28S，18S and 5S bands after ethidium bromide staining.The total RNA concentration was quantified by measuring absorbance at 260nm.cDNA was synthesized from RNA by the reverse transcription of 0.5μg of total RNA using the Rever AidTM First Strand cDNA Synthesis Kit(Fermentas，Canada)in a total volume of 20μL.The reaction mixture was then incubated at 42℃for 1h，70℃for 5min.Realtime PCR was performed on the StepOnePlus® Real-Time PCR System of Applied Biosystems.The rattus'cDNA sequences of Bcl-2，Bax，Fas，Caspase-3，i NOS and GAPDH were obtained from GenBank，and then designed primers by Primer Express 3.0 software(ABI).The primer sequences were showed in Table 1.For quantitative real-time PCR，each reaction was run in triplicate and contained the following：3.2μL PlatinumⒸSYBRⒸGreen qPCR Super Mix-UDG(Invitrogen，U.S.A.)，0.6μL Forward primer and Reverse primer(10μm)，0.2μL ROX Refrence Dye(Invitrogen，U.S.A.)and 0.4μL cDNA template in a final reaction volume of 10μL.Cycling parameters were 50℃for 2min and 95℃for 2min to activate DNA polymerase，and then followed by 40 cycles of 95℃for 15s and annealing at 60℃for 30s.After amplification，the melting curve analysis was performed as 95℃for 15s，60℃for 30s and 95℃for 15s to confirm formation of the expected PCR products.Products from all assays were subjected to 2%agarose gels electrophoresis to confirm specificity.The emission data were quantified using the cycle threshold(C t)value.Data were normalized to GAPDH and presented as the mean fold change as compared to controls.
Table 1 Primer Sequences of Target Genes for Real Time RT-PCR(https://www.daowen.com)
