The total RNA was extracted (Biomed, Primer Script Reverse, China) and cDNA was synthesized with TaKaRa Kit (TaKaRa Biotechnology Co Ltd, Jan), DNA extraction Kit (Beijingzhili Biotech) was adopted to prepare the DNA for iNOS methylation detection, which was carried out with Kang Wei methylation kit (Beijing Kang Wei co Ltd., Beijing) according to its manual. The Primers for methylation detection were listed in Table 1 . The qPCR was performed according to the instructions. The cDNAs were used for real-time quantitative PCR with MaximaTMSYBR Green/ROX qPCR Master Mix (2×) in Mx3000P qPCR Systems (Agilent Technologies Stratagene Products Division, USA) to detect IL-6, TNF-α, MMP9, TIMP1, eNOS, iNOS and nNOS , and all primers for qPCR were shown in Table 2 .
Primer script reverse
Manufactured by Takara Bio
Sourced in China
Primer Script Reverse is a reverse transcriptase enzyme used for the synthesis of complementary DNA (cDNA) from RNA templates. It is designed for high-efficiency, sensitive, and accurate reverse transcription of RNA into cDNA for downstream applications such as RT-PCR and gene expression analysis.
Automatically generated - may contain errors
2 protocols using primer script reverse
1
Analyzing iNOS Methylation and Gene Expression
2
Quantitative PCR analysis of Nephrin gene
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Total RNA from CIHP-1 cells seeded into a 6-well plate (6x104 cells/well) was extracted using TRIzol® reagent (Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the PrimerScript reverse transcriptase (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. qPCR analysis was performed on a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR green Premix Ex Taq II (Qiagen GmbH), according to the manufacturer's protocol. The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 5 min; followed by 40 cycles of denaturation at 95 ˚C for 45 sec, annealing at 50˚C for 45 sec and elongation at 72˚C for 45 sec; and a final extension step at 72˚C for 10 min. GAPDH was used as an internal reference gene and the relative gene expression was determined using the 2-ΔΔCq method (24 (link)). The following primer pairs were used for qPCR: Nephrin forward, 5'-CTGCCTGAAAACCTGACGGT-3' and reverse, 5'-GACCTGGCACTCATACTCCG-3'; and GAPDH forward, 5'-TGTGGGCATCAATGGATTTGG-3' and reverse, 5'-ACACCATGTATTCCGGGTCAAT-3'.
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