Total RNA was extracted from samples by RNAiso Plus (TaKaRa, Cat. No. 9108) according to the manufacturer’s instructions. mRNA expression levels were quantified by quantitative real-time RT-PCR. The reverse transcription reaction was performed using 1 μg of total RNA with a HiScript II 1st Strand cDNA Synthesis Kit ( + gDNA wiper; Vazyme, Cat. No. R212). Quantitative real-time PCR was performed using ChamQ SYBR Color qPCR Master Mix (High ROX Premixed; Vazyme, Cat. No. Q441) at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. PCR was performed on an ABI Q6 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA), and changes in expression were calculated using the 2-∆∆Ct method. Primers are listed in Table S2 .
Chamq sybr color qpcr master mix high rox premixed
Manufactured by Vazyme
ChamQ SYBR Color qPCR Master Mix (High ROX Premixed) is a ready-to-use solution for real-time quantitative PCR (qPCR) analysis. It contains SYBR Green I dye, DNA polymerase, and other necessary reaction components.
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2 protocols using chamq sybr color qpcr master mix high rox premixed
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Quantitative RNA Expression Analysis
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Quantitative RNA Expression Analysis
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Total RNAs were extracted from Mkrn2-WT and Mkrn2-KO MEFs, as well as from 293T cells, and purified using TRIzol reagent (catalog# 15596018, Invitrogen) according to the manufacturer's instructions. RT-PCR was performed using the HiScript II Q RT SuperMix (+gDNA wiper) (catalog# R323, Vazyme Biotech, Nanjing, China). qRT-PCR was performed using ChamQ SYBR Color qPCR Master Mix (High ROX Premixed) (catalog# Q441, Vazyme Biotech Co., ltd.) on a 7900HT system. Gapdh, β-actin, and Hprt levels were used as internal controls,21 (link)22 (link) and relative quantification (2−ΔΔCt) was used to calculate fold changes. The primer sequences are listed in Supplementary Table 2
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