Total mRNA from macrophages was extracted with a FastPure cell/tissue total RNA isolation kit V2 (Vazyme). mRNA was reverse transcribed using the HiScript III RT supermix for quantitative PCR (qPCR) (+gDNA wiper) (Vazyme). qPCR analyses were performed using ChamQ universal SYBR qPCR master mix (Vazyme). mRNA expression was assessed by the comparative cycle threshold (CT) method (2−ΔΔCT); beta-actin genes (forward primer: 5’-GGCTGTATTCCCCTCCATCG-3’, revers primer: 5’-CCAGTTGGTAACAATGCCATGT-3’) were used as housekeeping genes. TNFA genes (forward primer: 5’-CCCTCACACTCAGATCATCTTCT-3’, revers primer: 5’-GCTACGACGTGGGCTACAG-3’) and iNOS genes (forward primer: 5’-GTTCTCAGCCCAACAATACAAGA-3’, revers primer: 5’-GTGGACGGGTCGATGTCAC-3’) are used as markers of M1 macrophages.
Hiscript 3 rt supermix for quantitative pcr qpcr gdna wiper
Manufactured by Vazyme
Sourced in China
HiScript III RT supermix for quantitative PCR (qPCR) (+gDNA wiper) is a reagent designed for reverse transcription and quantitative PCR. It includes a reverse transcriptase enzyme, reaction buffer, and a genomic DNA removal component.
Automatically generated - may contain errors
2 protocols using hiscript 3 rt supermix for quantitative pcr qpcr gdna wiper
1
Quantification of M1 Macrophage Markers
2
qPCR Analysis of Hippocampal miRNA in T2DM Rats
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Total RNA was isolated from the hippocampal tissue of rats in the control and T2DM groups using RNAiso Plus reagent (Takara, Dalian, China) and then reverse transcribed into cDNA using HiScript III RT SuperMix for quantitative PCR (qPCR) (+g DNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China). Real-time qPCR was performed using miRNA Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) on a BioRad qPCR system with the following primer pair: 5’-TGCATGGTGACTGCTACCTTCTC-3’, 5’-AAATCACAGCAGCCTACCCACTC-3’. The PCR conditions started with a denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94°C for 25 s, annealing at 50.5 °C for 40 s, and extension at 72 °C for 30 s, ending with a final extension step at 72 °C for 5 min. RNA relative expression was calculated by the 2-ΔΔCt method with reduced glyceraldehyde-phosphate dehydrogenase as the control.
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