The total RNA from jejunums of mice and FHs74Int cells was extracted with HiPure Total RNA Mini Kit (Magen, Guangzhou, China) and subjected to cDNA synthesis with Hifair II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit (Yeasen Biotechnology, Shanghai, China). The qPCR was performed with Hifair qPCR SYBR Green Master Mix (No Rox) kit (Yeasen Biotechnology) on a Roche 480 Light Cycler. The primers used for PCR amplification are shown as follows: 5′-ACTGGCCGACCTAAGGGAG-3′, 5′-GCCAAAGGCAAGTAGCCAATA-3′ (ACSL4) and 5′-CTGGGACGACATGGAGAAAA-3′, 5′-AAGGAAGGCTGGAAGAGTGC-3′ (ACTB). ACTB was used as a normalizing control. The fold changes of mRNA were calculated with the 2−ΔΔCt method.
Hifair 2 1st strand cdna synthesis supermix for qpcr gdna digester plus kit
Manufactured by Yeasen
Sourced in China
The Hifair II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit is a laboratory reagent designed for the synthesis of first-strand cDNA from RNA samples. The kit includes a proprietary enzyme blend that performs both reverse transcription and genomic DNA (gDNA) digestion in a single reaction, allowing for efficient cDNA synthesis while removing potential gDNA contamination.
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2 protocols using hifair 2 1st strand cdna synthesis supermix for qpcr gdna digester plus kit
1
Quantifying ACSL4 Expression in Intestinal Cells
2
Quantifying MSH4 Expression in Transfected Cells
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After 48 h of transfection with wild-type and mutant expression plasmid, cell samples were collected. Total RNA was extracted using Trizol (TaKaRa, Japan) and cDNA was synthesized using the Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit (Yeasen, Shanghai, China) according to the manufacturer’s protocol. Quantification of gene expression was performed using qPCR with the ABI Prism 7500 (Applied Biosystems). The cDNA was used as template in qPCR reactions with specific primers and probes for MSH4, as well as GAPDH as control. Thermal cycling conditions consisted of an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were analyzed using the 2^(-ΔCt) method, and significance was determined using a t-test with a significance level of p < 0.05. The primers used are listed in Supplementary Table 1.
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