Purified RNA was applied as a template to synthesize the first-strand cDNA based on a relevant RT Reagent Kit with genomic DNA Eraser (Takara, Japan). After reverse transcription, gene expression was quantified in a RT-PCR system (Roche 9000, Switzerland) via a EX Taq kit (Takara, Tokyo, Japan) according to the manufacturer’s protocols. All reactions were conducted in triplicate. Corresponding specific primers (Table S2 ) were designed using Primer Premier 6.0. β-Actin was regarded as internal reference and relative gene levels were calculated based on 2-ΔΔCt method.
Rt reagent kit with genomic dna eraser
Manufactured by Takara Bio
Sourced in Japan, China
The RT Reagent Kit with genomic DNA Eraser is a laboratory product designed for RNA reverse transcription. It contains the necessary reagents to convert RNA into complementary DNA (cDNA) for further analysis.
Automatically generated - may contain errors
2 protocols using rt reagent kit with genomic dna eraser
1
Quantitative RT-PCR Gene Expression Analysis
2
Quantitative Real-Time PCR for Gene Expression
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Total RNA was isolated with TRIzol® Reagent (Thermo Fisher Scientific, USA). RNA integrity was determined by RNA formaldehyde denaturing gel electrophoresis. RNA concentration and purity was measured by Nanodrop2000 (Thermo Fisher Scientific, America), traces of DNA were removed and reverse transcription PCR was executed by RT reagent Kit with genomic DNA Eraser (Takara Bio, China).
The mRNA expression levels of target genes were measured by qRT-PCR with the SYBR Premix Ex Taq™ II (Takara Bio, China) on a Thermo Fisher Scientific Q7 system following the manufacturer’s directions. The qRT-PCR primers specific for the target genes can be found as Supplementary TableS1 . All primers’ specificity was assessed by melting curve analysis. The qRT-PCR was performed following the next procedure: 95 °C for 2 min and then 40 cycles at 95 °C for 15 s, 59 °C for 20 s and 72 °C for 20 s. Changes in mRNA levels of target genes were expressed as the fold of induction relative to the negative control.
The mRNA expression levels of target genes were measured by qRT-PCR with the SYBR Premix Ex Taq™ II (Takara Bio, China) on a Thermo Fisher Scientific Q7 system following the manufacturer’s directions. The qRT-PCR primers specific for the target genes can be found as Supplementary Table
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