Total RNA, extracted from colonic tissues by RNAsimple Total RNA Kit (Tiangen, Beijing, China), were reverse transcribed into cDNA using Hifair™ cDNA Synthesis Kit (Yeasen, Shanghai). Real-time quantitative PCR analysis was performed by SYBR® Green Realtime PCR Master Mix (Yeasen) plus 7500 Fast Real-Time PCR System (Applied Biosystems, Foster city, CA, USA) with gene-specific primers (Supporting Information Table S2 ). The expression level of genes was normalized to the internal housekeeping genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for human samples or β-actin for mouse samples].
Hifair cdna synthesis kit
Manufactured by Yeasen
Sourced in China
The Hifair™ cDNA Synthesis Kit is a laboratory tool designed for reverse transcription of RNA to complementary DNA (cDNA). The kit includes the necessary reagents and enzymes to perform this process.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green real time pcr master mix, by Yeasen (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rnasimple total rna kit, by Tiangen Biotech (2 mentions)
Hifair cdna synthesis supermix kit, by Yeasen (1 mentions)
Trizol method, by Thermo Fisher Scientific (1 mentions)
Trizol solution, by Tiangen Biotech (1 mentions)
Nanodrop microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
4 protocols using hifair cdna synthesis kit
1
Real-Time qPCR Analysis of Gene Expression
2
RNA Extraction and cDNA Synthesis for G. elegans
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The G. elegans samples were ground to extract total RNA using the TRIzol method (Invitrogen, USA). According to the Hifair® cDNA Synthesis Kit (Yeasen, Shanghai, China), RNA from G. elegans leaves was reverse-transcribed into cDNA for subsequent gene cloning experiments. Based on the Hifair® cDNA Synthesis SuperMix kit (Yeasen, Shanghai, China), RNA of G. elegans tissues and G. elegans samples under different treatments was reverse-transcribed into cDNA and used as a template for qRT-PCR analysis.
3
Quantitative Real-Time PCR Analysis of Human Tumor Tissues
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Total RNA from human tumor tissues was extracted using the RNAsimple Total RNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instruction. The tissues were homogenized in the TRIzol solution (Tiangen). The concentration of total RNA was determined using NanoDrop Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific) and subsequently reverse transcribed into cDNA (500 ng per unit) using Hifair™ cDNA Synthesis Kit (Yeasen, Shanghai) with consecutive incubation of 25°C for 5 min, 42°C for 30 min. and 85°C for 5 min. Quantitative real-time PCR was performed with the cDNA and the specific primers using SYBRⓇ Green Realtime PCR Master Mix (Yeasen) by 7500 Fast Real-Time PCR System (Applied Biosystems, Foster city, CA, USA) for PCR amplification with holding stage (95°C for 30 s), cycling stage (95°C for 15 s and 60°C for 34 s for 40 cycles). GAPDH was used as the internal housekeeping gene for human samples. The sequences of used primers were listed in Table S2.
4
Quantification of FtZIP Transcripts via qRT-PCR
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The expression levels of FtZIP transcripts were analyzed using quantitative real-time PCR (qRT-PCR) assay. Total RNA was extracted from the roots (7-d seedlings), stems (10-d seedlings), leaves (10-d seedlings), flowers, fruits and seeds using an RNAprep Pure Plant Kit (Tiangen, Beijing, China). Then, cDNA synthesis was performed in a 20 μl reaction mixture containing 1 μg of total RNA and a mixture of Hifair® cDNA Synthesis Kit (Yeasen, Shanghai, China). The real-time PCR mixture contained 1 μl cDNA, 1 μl forward and reverse primers, and 10 μl 2 x SYBR Green (TaKaRa, Beijing, China). The qRT-PCR was performed using a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, Hercules, California, CA, USA). All reactions were performed in three triplicates with the following cycling conditions: 95°C for 3 min; 30 cycles each at 95°C for 10 s and 56°C for 30 s, and 72°C for 20 s. The 2−ΔΔCt method was used for the analysis of qRT-PCR (Livak and Schmittgen, 2001 (link)). The housekeeping gene FtH3 (ID: HM628903) was used as an internal control (Li C et al., 2019 (link)). All primers are shown in Supplementary Table S1
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