Total mRNA was isolated from tissues or cells using Trizol reagent (#15596026, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription was performed in a final volume of 20 μL containing 1 μg RNA using the HiScript II select qRT supermix (R222-01, Vazyme, Jiangsu, China). qRT-PCR was performed using gene specific primer sets and SYBR Green (Q311-03, Vazyme) on a qRT-PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). All primers were designed by ourselves and synthesized in the Generay Biotech Co., Ltd. (Shanghai, China). The reaction conditions were 95 °C for 30 s, 40 cycles of 95 °C for 10 s, 56 °C for 30 s and 72 °C for 30 s. Relative gene expression was calculated using the ΔΔCt method with β-actin as a reference gene. Primer sequences were shown in Supporting Information Table S1 .
Hiscript 2 select qrt supermix
Manufactured by Vazyme
Sourced in China
HiScript II Select qRT SuperMix is a reverse transcription and quantitative real-time PCR (qRT-PCR) reagent kit designed for the detection and quantification of RNA targets. It provides efficient and sensitive RNA-to-cDNA conversion and real-time PCR amplification in a single reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Qrt pcr detection system, by Bio-Rad (1 mentions)
Sybr green, by Vazyme (1 mentions)
Applied biosystems real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix, by Vazyme (1 mentions)
Cfx manager 3.1 system, by Bio-Rad (1 mentions)
Hiscript 2 q select rt supermix for qpcr, by Vazyme (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
4 protocols using hiscript 2 select qrt supermix
1
Quantitative RT-PCR for Gene Expression Analysis
2
Quantitative Real-Time PCR Analysis of Gene Expression
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Using the PrimeScript RT reagent kit, 1 μg of RNA isolated from the RAW 264.7 cells was reverse-transcribed to cDNA with TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and then amplified using SYBR green Premix Ex Taq II (Vazyme Biotech, Nanjing, China) with HiScript II Select qRT SuperMix (Vazyme Biotech) in an Applied Biosystems Real-Time PCR System (Life Technologies) under the following reaction conditions: initial denaturation, 95°C, 30 seconds; 40 cycles of 95°C, 5 s and 60°C, 30 second; annealing, 95°C, 15 seconds, 60°C, 1 minute, and 95°C for 15 seconds. The ΔCT method was adopted for data analysis, and actin was used as the reference gene for calculating the relative gene expression levels of the treatment groups. Table 1 shows the list of qPCR primers used in this study.
3
Hippocampal mRNA Expression Quantification
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The total RNA was extracted from the rats’ hippocampal tissue using TRIzol reagent (Invitrogen, USA) and converted into cDNA using HiScript II Select qRT SuperMix (Vazyme, China) according to the supplier’s instructions. The cDNA products were kept at −20 °C prior to use. The mRNA levels were quantified in triplicate by RT-qPCR with SYBR premix (Vazyme, China) on a CFX Manager 3.1 system (Bio-Rad, USA). The mRNA levels were evaluated by the 2-△△Ct method. Gapdh was chosen as the internal reference genes [93 (link)]. The primers used for RT-qPCR were presented in Supplementary Table S2 .
4
RNA Extraction and RT-qPCR Quantification
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The extraction and quantification of total RNA were processed as previously described. Later, 1 μg total RNA was converted into cDNA using the HiScript® II Q Select RT SuperMix for qPCR (R232-01; Vazyme, Nanjing, China) according to the manufacturer's protocol−4 μl 5 × HiScript II Select qRT SuperMix, 1 μl random hexamers (50 ng/μl), and 1 μg RNA, which were run at 37°C for 15 min and 85°C for 2 min. Subsequently, the RT-qPCR reaction was performed using AceQ® qPCR (Q131-01; Vazyme) in a protocol that is to add 1 μl cDNA to 4 μl master mix, including 2.5 μl SYBR® Green Master Mix (Low ROX Premixed; Q131-01; Vazyme), 0.2 μl reverse and forward primers, and 1.1 μl diethylpyrocarbonate water. Then, qPCR was performed under the conditions which included an initial step at 95°C for 5 min, 40 cycles at 95°C for 10 s, and at 60°C for 30 s, and final extension at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s, with a LightCycler 480 II (Roche). The primer sequences designed through Primer Premier 6 are listed in Table 1 . GAPDH is used as the internal control. So far, 10 blood samples from newborns were used to perform RT-qPCR validation.
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