Revertaid first strand cdna synthesis kit

Total RNA extraction from renal tissue specimens and NRK-52E cells was carried out with TRIzol reagent (Invitrogen, U.S.A.). A RevertAid™ First Strand cDNA Synthesis Kit (Thermo, U.S.A.) was employed for cDNA production. Subsequently, Talent qPCR PreMix (SYBR Green) (Tiangen, Beijing, China) was employed for qPCR. The corresponding primer sequence is shown in Table 1. Reverse transcription and quantitative detection of miR-27a were performed based on the protocols included in the RevertAid™ First Strand cDNA Synthesis Kit and Bulge-Loop™ miRNA qRT-PCR Primer Kit (RiboBio, Guangzhou, China), respectively. Relative quantification was carried out with the 2^−ΔΔCt method; U6 or β-actin served as an internal control.

microRNAs were enriched separately from Larger RNAs (>200nt) using the miRNeasy Micro Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The concentration and integrity of RNA were assessed using a NanoDrop 2000 spectrophotometer (thermo fisher scientific, New York, NY, USA). The miRNA was reverse transcribed into complementary DNA (cDNA) using a Thermo Scientific RevertAid First Strand cDNA Synthesis Kit with a specific RT primer (RiboBio, Guangzhou, China). The PCR was carried out in an ABI 7500 Sequence Detection System (ABI, Life Technologies, New York, USA) using SYBR Green Master Mix (Life Technologies, New York, NY, USA), and the relative level of expression was normalized to U6 or β-actin. Each sample was analyzed in triplicate. Data analysis was performed using the 2^−ΔΔCT method. The primer was shown in supplementary Table 1.