2.7. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Detection of Gene mRNA Expression

Forward and reverse primer sequences were designed using Primer 5.0 online software (Table 1). The primer sequences were synthesized from GENIWIZ Technologies. Total RNA was extracted from renal tissue using TRIzol reagent (Invitrogen). Reverse transcription technology was used to turn RNA into cDNA. The conditions of reverse transcription reaction system were as follows: 37°C 15 min ∗3(Reverse transcription reaction), 85°C 5 sec (Reverse transcriptase inactivation reaction), and 4°C. Gene expression levels were calculated using Roche LightCycler 480. The conditions of the amplification reaction system were as follows: predenaturation at 95°C for 30 sec, 40 cycles at 95°C for 5 sec, and 60°C for 34 sec. ΔCt = target gene Ct – GAPDH gene Ct, ΔΔCt = Ct in the experiment group - Ct in the control group. The value of Ct is the fluorescence value of amplified circulating gene. Target gene relative expression was quantified using 2-ΔΔCt method. The data was analyzed by three independent experiments and the relative mRNA expression was normalized with GAPDH as an internal reference.

List of primer sequences for reverse transcription-quantitative polymerase chain reaction.