The third instar nymphs of the SS and RS (F20) strains were collected, and each strain was divided into three groups with 30 insects in each group. The total RNA was extracted using the RNA-esay™ Isolation Reagent (Vazyme Biotech Co., Ltd, Nanjing, China). After that, reverse transcription of RNA to first-strand cDNA was performed using HiScript®III RT SuperMix for qPCR (+gDNA wiper) reagent kit (Vazyme Biotech Co., Ltd, Nanjing, China). The qRT-PCR reaction was carried out using ChamQ universal SYBR® qPCR Master Mix reagent kit (Vazyme Biotech Co., Ltd, Nanjing, China). The primers used in qRT-PCR analyses were provided in Supplementary Table S1 , and the level of P450 gene transcripts was normalized to that of GAPDH. Relative quantification was performed using the 2−ΔΔCTmethod (Livak and Schmittgen, 2021 (link)).
Hiscript 3 rt supermix for qpcr gdna wiper reagent kit
Manufactured by Vazyme
Sourced in China
HiScript®III RT SuperMix for qPCR (+gDNA wiper) is a reagent kit designed for reverse transcription and real-time quantitative PCR (qPCR) analysis. The kit includes a reverse transcriptase enzyme and a gDNA wiper component to remove genomic DNA contamination.
Automatically generated - may contain errors
Lab products found in correlation
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
Chamq universal sybr qpcr master mix reagent kit, by Vazyme (1 mentions)
Lightcycler 480 2 real time pcr system, by Roche (1 mentions)
Spin column plant total rna purification kit, by Sangon (1 mentions)
Mir x mirna first strand synthesis kit, by Takara Bio (1 mentions)
Aceq qpcr sybr green master mix high rox premixed kit, by Vazyme (1 mentions)
Total rna extraction reagent, by Vazyme (1 mentions)
Easyspin plus plant rna kit, by Aidlab (1 mentions)
Lightcycler 480 2 pcr system, by Roche (1 mentions)
4 protocols using hiscript 3 rt supermix for qpcr gdna wiper reagent kit
1
P450 Gene Expression Analysis in Insect
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated using the Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China). After DNase I digestion, total RNA was used for reverse transcription (RT) using the HIScript® III RT SuperMix for qPCR (+ gDNA wiper) reagent kit (Vazyme Biotech, Jiangsu, China). PCR reaction mixture was prepared using the ChamQTM Universal SYBR® qPCR Master Mix (Vazyme Biotech) and then loaded onto LightCycler® 480 II Real-Time PCR System (Roche) for amplification with the following conditions: 30 s at 95 °C, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The specificity of amplified products was monitored by melting curve analysis and verified by agarose gel electrophoresis. Gene-specific primers used in this study were listed in Supplementary Table S1 . β-tubulin (AtTUB4) was used as an internal control to calculate the relative expression levels of target genes using the 2−ΔΔCq method.
3
Quantifying RNA Expression in Mouse Hippocampus
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Total RNA was purified from the hippocampus of mice and HT22 cells using total RNA extraction reagent (Vazyme, Nanjing, China) according to the manufacturer’s protocol. Total cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) reagent kit (Vazyme, Nanjing, China). The Mir-X miRNA first strand was synthesized with the Mir-X miRNA First-Strand Synthesis Kit (Takara Bio USA, Mountain View, CA, USA). qRT-PCR was performed using an AceQ qPCR SYBR Green Master Mix (High ROX Premixed) kit (Vazyme, Nanjing, China) on a Quantagene q225 Real-Time PCR system (kubo Technology Ltd., Beijing, China) according to the manufacturer’s protocol. The relative expression of mRNA or miRNA in each sample was normalized to β-actin (mRNA) or U6 (miRNA) by using the 2−ΔΔCt method. The primer sequences are listed in Table S2 .
4
Quantitative Expression Profiling of Cotton
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Total RNA was isolated using the EASYspin Plus Plant RNA Kit (Aidlab, Beijing, China) following manufacturer’s instructions. Reverse transcription (RT) was performed using the HIScript® III RT SuperMix for qPCR (+gDNA wiper) Reagent Kit (Vazyme Biotech, Nanjing, China). A quantitative PCR analysis was carried out using the ChamQTM Universal SYBR® qPCR Master Mix (Vazyme Biotech) and gene-specific primers (Supplementary Table S1 ) in a LightCycler® 480II PCR system (Roche, Basel, Switzerland). The cycling conditions were 30 s at 95 °C, 40 cycles of 10 s at 95 °C, and 30 s at 60 °C. The specificity of amplified products was monitored by melting curve analysis, and verified by agarose gel electrophoresis. Relative expression levels of genes were normalized to GhUBQ7 as an internal control and calculated using the 2−∆∆Cq method. When necessary, the house-keeping gene GhACT7 was used for plate-to-plate equilibration.
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