The expression of cyp51A, cyp51B, AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, cdr1b, mfs56, and m85 was determined by real-time qPCR. The ΔAfu-emi1, WT, and ΔAfu-emi1::Afu-emi1+ strains were cultured on SDA for 2 days. Total RNA was extracted using the TRIeasy LS total RNA extraction reagent (Yeasen Biotechnology) and transcribed to cDNA via the Hifair II first-strand cDNA synthesis supermix for qPCR (gDNA Digester Plus) (Yeasen, Biotech Shanghai, China) according to the manufacturer’s instructions. The RT-qPCR was performed by using Hieff qPCR SYBR green master mix (Yeasen Biotechnology) in an ABI 7500 reverse transcription real time-PCR system. The primers used for RT-qPCR are shown in Table S2 in the supplemental material. The levels of each gene were calculated with the 2−ΔΔCT method (26 (link)). The relative expression levels were normalized to the level of the internal control actin gene, where ΔCT of target gene = CT of target gene − CT of actin. Experiments were performed in triplicate.
Hifair 2 first strand cdna synthesis supermix for qpcr gdna digester plus
Manufactured by Yeasen
Sourced in China
Hifair II first-strand cDNA synthesis supermix for qPCR (gDNA Digester Plus) is a laboratory product designed for the synthesis of complementary DNA (cDNA) from RNA samples. It includes a reagent mix for the conversion of RNA into cDNA, which can then be used for quantitative real-time PCR (qPCR) analysis. The product also contains a gDNA Digester component to remove any genomic DNA contamination from the RNA samples.
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Lab products found in correlation
Hieff qpcr sybr green master mix, by Yeasen (1 mentions)
Trieasy ls total rna extraction reagent, by Yeasen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Hieff unicon qpcr sybr green master mix, by Yeasen (1 mentions)
2 protocols using hifair 2 first strand cdna synthesis supermix for qpcr gdna digester plus
1
Transcriptional Analysis of Antifungal Resistance Genes
2
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from tissues and cell lines with TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The quality and quantity of isolated RNA were detected. cDNA was synthesized from approximately 1 μg of RNA from each sample using Hifair II first-strand cDNA synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen Biotech). Total DNAs from cells were extracted using an AxyPrep multisource gDNA miniprep kit (Axygen, USA). qRT-PCR was performed on a LightCycler 480 real-time PCR system (Roche Applied Science, Germany) using Hieff Unicon qPCR SYBR Green master mix (Yeasen Biotech). The relative mRNA expression or DNA content was calculated using the ΔΔCt method. The primers are listed in Table S2 .
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