The total RNA was extracted using TRIzol (TaKaRa, China) according to the standard procedures. The RNA was used as template for reverse transcription to synthesize cDNA using HiScript III RT SuperMix reagent (Vazyme, China). The RT-qPCR was performed according to the following conditions using the AceQ qPCR SYBR Green Master Mix (Vazyme, China): initial denaturation at 95 ℃ for 5 minutes, followed by 40 cycles at 95 ℃ for 10 seconds and 60 ℃ for 30 seconds, finally at 95 ℃ for 15 seconds, 60 ℃ for 60 seconds and 95 ℃ for 15 seconds. The PCR reaction was monitored on ABI StepOneplusTM Real Time PCR System (ThermoFisher, USA). The GAPDH gene was used as internal reference to normalize the results and mRNA expression was calculated through relative quantification (2−ΔΔCT). The primer sequences were designed using Primer-BLAST. The primer sequences were as follows: GAPDH, forward 5'-GAGAAGGCTGGGGCTCATTT, reverse 5'-AGTGATGGCATGGACTGTGG. PD-L1, forward 5'-CAGCAACTTCAGGGGGAGAG, reverse 5'-TTTGCGGTATGGGGCATTGA. TGF-β1, forward 5'-AGCTGCGCTTGCAGAGATTA, reverse 5'-AGCCCTGTATTCCGTCTCCT. E-Cadherin (CDH1), forward 5'-AACCCAAGCACGTATCAGGG, reverse 5'-ACTGCTGGTCAGGATCGTTG. Fibronectin 1 (FN1), forward 5'-ATGAGAAGCCTGGATCCCCT, reverse 5'-CAGTTGGGGAAGCTCATCTGT.
Hiscript 3 rt supermix reagent
Manufactured by Vazyme
Sourced in China
HiScript III RT SuperMix is a reverse transcription reagent for the synthesis of first-strand cDNA from total RNA or poly(A)+ RNA. It contains a high-performance reverse transcriptase, RNase inhibitor, and other essential components for efficient cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Trizol, by Takara Bio (1 mentions)
Sybr qpcr master mix, by Vazyme (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nd 2000 micro spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Total rna extraction reagent, by Vazyme (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
3 protocols using hiscript 3 rt supermix reagent
1
Quantitative Analysis of RNA Expression
2
Real-Time PCR Analysis of Gene Expression
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Total RNA was extracted from the ileum (mucosal) and liver samples using the Total RNA Extraction Reagent (Vazyme Biotechnology, Nanjing, Jiangsu, China) and quantified using an ND-2000 micro spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After the determination of RNA quality and concentration, 1 µg of total RNA was reverse-transcribed into complementary DNA (cDNA) using the HiScript III RT SuperMix Reagent (Vazyme Biotechnology), following the manufacturer’s instructions. The mRNA expression levels of specific genes were quantified via real-time polymerase chain reaction (PCR) using the SYBR qPCR Master Mix (Vazyme Biotechnology) and the QuantStudio 5 Real-Time PCR System (Thermo Scientific, Wilmington, DE, USA). The SYBR Green PCR reaction mixture consisted of 10 µL TB Green Premix Ex Taq, 0.4 µL ROX Reference Dye II, 2 µL cDNA template, 0.4 µL of each primer (total 0.8 µL, 10 µmol/L) and 6.8 µL of double-distilled H2O. The reaction conditions were as follows: pre-run at 95 °C for 30 s, 40 denaturation cycles at 95 °C for 10 s and annealing at 60 °C for 30 s. Each sample was run in triplicate. The relative mRNA expression levels were analyzed via the 2−ΔΔCt method after normalization against β-actin, and the results displayed a similar trend when GAPDH served as the housekeeping gene.
3
RNA Isolation, cDNA Synthesis, and RT-qPCR
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Total RNA was isolated using RNAiso Plus reagent (#9109, Takara), and cDNA was synthesized using HiScript III RT SuperMix reagent (#R323-01, Vazyme). Real-time quantitative PCR (RT-qPCR) was carried out by ChamQ Universal SYBR qPCR Master Mix (#Q711-03, Vazyme). The primers used for RT-qPCR are provided in Supplementary Table S5.
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