Total RNA was extracted from HeLa and 293T cells using the Trizol method (RNAiso PLUS, TaKaRa). The concentrations and purity of RNA were determined by UV spectrophotometry, and cDNA was synthesized by transcription kit (HifairTM 1st Strand cDNA Synthesis SuperMix for qPCR, YEASEN). The PCR products were identified by 2% agarose gel electrophoresis and verified by sequencing.
Hifair 1st strand cdna synthesis supermix for qpcr
Manufactured by Yeasen
Sourced in China
Hifair® 1st Strand cDNA Synthesis SuperMix for qPCR is a ready-to-use reagent kit designed for the reverse transcription of RNA into cDNA for real-time quantitative PCR (qPCR) applications. The kit includes all necessary components, including reverse transcriptase, RNase inhibitor, and reaction buffer, in a single tube format for convenient and efficient cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Hieff qpcr sybr green master mix, by Yeasen (6 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Hieff unicon universal blue qpcr sybr green master mix, by Yeasen (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Transzol up plus rna kit, by Transgene (1 mentions)
Cfx connect optics module, by Bio-Rad (1 mentions)
Lightcycler 96, by Roche (1 mentions)
9 protocols using hifair 1st strand cdna synthesis supermix for qpcr
1
RNA Extraction and cDNA Synthesis
2
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from the HEK293T and HeLa cells using the TRIzol reagent (RNAiso PLUS, TaKaRa Bio Inc., Shiga, Japan). RNA concentration and purity were assessed using UV spectrophotometry, and the cDNA was synthesized using HifairTM 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus, YEASEN Biotechnology, Shanghai, China). The PCR products were identified using 2% agarose gel electrophoresis and verified by sequencing.
3
Quantitative Gene Expression Analysis
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The samples of wild-type worms, liver, and kidney were prepared, and their RNA was extracted from them using the Total RNA Kit (OMEGA, BioTek). Then, reverse transcription was conducted using Hifair® 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen, 11123ES10) by LongGene A200. Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen, 11184ES03) was used to perform qPCR (BIO-RAD, CFX96). Three replicate experiments were conducted. An unpaired t-test was used to calculate the P values, and error bars represented SEM.
4
Quantitative Real-Time PCR of Immune Markers
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By the method of TRIzol, total RNA from mice bone marrow cells, peripheral blood cells and intestines were extracted. Reverse transcription was conducted by using of Hifair® 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen Biotechnology, Shanghai, China). qPCR was performed through Roche LightCycler® 96 System (Roche, Shanghai, China) with the use of Hieff® qPCR SYBR® Green Master Mix (Yeasen Biotechnology, Shanghai, China). Actin was used as the internal reference in the whole investigation. Primers were either synthesized by using the Primer Premier 5.0 program or supported by previous studies. The sequences were shown as follows:
CD11b, 5′-CAATAGCCAGCCTCAGTGC-3′ (forward) and 5′-GAGCCCAGGGGAGAAGTG-3′ (reverse);
Ly-6C, 5′-GCAGTGCTACGAGTGCTATGG-3′ (forward) and 5′-ACTGACGGGTCTTTAGTTTCCTT-3′ (reverse);
F4/80, 5′-ACCTAGACATCGAAAGCAAA-3′ (forward) and 5′-TGATTATGAAACAGCCAACA-3′ (reverse);
CCR2, 5′-GAGTGAGAAGGAGGAGATATGC-3′ (forward) and 5′-AACACAGATAGGAGAAGGAACC 3′ (reverse);
Actin, 5′-GCTGTCCCTGTATGCCTCTG-3′ (forward) and 3′-TGATGTCACGCACGATTTCC-5′ (reverse).
CD11b, 5′-CAATAGCCAGCCTCAGTGC-3′ (forward) and 5′-GAGCCCAGGGGAGAAGTG-3′ (reverse);
Ly-6C, 5′-GCAGTGCTACGAGTGCTATGG-3′ (forward) and 5′-ACTGACGGGTCTTTAGTTTCCTT-3′ (reverse);
F4/80, 5′-ACCTAGACATCGAAAGCAAA-3′ (forward) and 5′-TGATTATGAAACAGCCAACA-3′ (reverse);
CCR2, 5′-GAGTGAGAAGGAGGAGATATGC-3′ (forward) and 5′-AACACAGATAGGAGAAGGAACC 3′ (reverse);
Actin, 5′-GCTGTCCCTGTATGCCTCTG-3′ (forward) and 3′-TGATGTCACGCACGATTTCC-5′ (reverse).
5
Quantitative analysis of BnaGPAT9 expression
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Total RNA of ‘Zhongshuang 11’ was extracted using a TransZol Up Plus RNA Kit (Transgene, Shenzhen, China) and detected using a NanoDrop™ One (Thermo Fisher, Waltham, MA, USA). cDNA was synthesized using Hifair® 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, Shanghai, China). The expression patterns of BnaGPAT9-A1/C1 in roots, stems, leaves, and developing seeds (15, 20, 25, 30, 35, and 40 days after flowering) were determined by CFX Connect™ Optics Module (Bio-Rad, Hercules, CA, USA) with three biological replications. The 20-µl qRT‒PCR contained 10 µl of 2 x Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China), 0.004 nM forward primer, 0.004 nM reverse primer, and 9.2 µl of cDNA. The reaction program was initiated by predenaturation at 95°C for 5 min, and this step was followed by 40 cycles of denaturation (95°C for 10 s) and annealing (55°C for 30 s). The reference gene was ubiquitin-conjugating enzyme 9 (accession no. XM_013800933) [9 (link)], which was used to normalize the expression levels of BnaGPAT9-A1/C1. To distinguish BnGPAT9-A1/C1 by qPCR, primers were designed for the 3’ UTR and 5’ UTR differential regions of the BnaGPAT9-A1 and BnaGPAT9-C1 mRNA sequences, respectively. All primers are listed in Supplementary Table S4 .
6
RT-qPCR Gene Expression Analysis
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For RT-qPCR analysis, 1 μg of total RNA was reverse transcribed into cDNA using Hifair 1st Strand cDNA Synthesis SuperMix for qPCR (11141ES10; Yeasen Biotech, China) following the manufacturer’s instructions. Quantitative PCR was performed on a Lightcycler 96 (Roche) with Hieff qPCR SYBR Green Master Mix (No Rox) (11201ES03; Yeasen Biotech). Relative mRNA expression levels were calculated using the 2–ΔΔCT method and normalized to those of GAPDH. The sequences of all the primers used in this study are shown in Supplementary Table 1 .
7
Quantification of Stress-Induced Gene Expression
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Total RNA was isolated from leaf tissues of TM-1 under salt stress treatment at 0 h, 1 h, 3 h, 6 h and 12 h using RNAprep pure Plant Kit (code: DP432, Tiangen, Beijing, China). First-strand cDNA was synthesized using Hifair 1st Strand cDNA Synthesis SuperMix for qPCR (YEASEN Biotech, Shanghai, China). The qRT-PCR was performed using Hieff qPCR SYBR GreenMaster Mix (YEASEN Biotech, Shanghai, China). The cotton ubiquitin gene UBQ7 was used as the internal control for the relative expression calculation. RNA isolation and qRT-PCR was manipulated based on the manufacturer’s instructions. The primers used for qRT-PCR were listed in Supplementary Table 2
8
Quantitative RT-PCR Analysis of Cotton Transcripts
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Total RNA was extracted from 0.10 g of cotton PR samples using RNAprep pure Plant Kit (code: DP432, Tiangen, Beijing, China) based on the instructions of the manufacturer. Reverse transcription was performed using Hifair 1st Strand cDNA Synthesis SuperMix for qPCR (YEASEN Biotech, Shanghai, China) with the same amount of total RNA (1μg). The gDNA digester mix was used to remove gDNA contamination. The qRT-PCR was performed using Hieff qPCR SYBR GreenMaster Mix (YEASEN Biotech, Shanghai, China). The reaction parameters were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 sec. The cotton ubiquitin gene UBQ7 was used as the internal control for each sample. The relative expression levels were calculated using the 2−ΔΔCt method. Primers for qRT-PCR are listed in Supplementary Table 1 , and one-way ANOVA statistical analysis was then performed.
9
Metabolomic and Transcriptomic Profiling
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We used Compound Finder 3.0 (CD 3.0, Thermo Fisher) to process the raw data files. Data of total spectral intensity normalized by the peak intensity was used to predict molecular formulas based on addition ions,fragment ions and molecular ion peaks. The mzCloud (https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) databases were used for accurate qualitative and relative quantitative results. Metabolites whose variable importance in projection (VIP) was ≥1 with p less than 0.05 for the model variables were defined as differentially accumulated metabolites.
Validation of RNA-Seq data by qRT-PCR
To validate the RNA-seq data, we examined the expressions of 13 candidate genes by qRT-PCR. The cDNA was synthesized using the Hifair ® 1st Strand cDNA Synthesis SuperMix for qPCR (11123ES60, YEASEN, China). The analysis of qRT-PCR was carried out using Hieff ® qPCR SYBR Green Master Mix (11202ES08, YEASEN, China). The qRT-PCR cycle parameters were set at 95 for 2 min, 95 for 10 s, and annealing temperature at 60 for 40 s, which comprised a total of 40 cycles. Then the 2 -CT method was used to calculate the relative induction ratios of treated samples compared with controls (Livak and Schmittgen, 2001). Three biological replications were used and three measurements were performed on each replicate.
Validation of RNA-Seq data by qRT-PCR
To validate the RNA-seq data, we examined the expressions of 13 candidate genes by qRT-PCR. The cDNA was synthesized using the Hifair ® 1st Strand cDNA Synthesis SuperMix for qPCR (11123ES60, YEASEN, China). The analysis of qRT-PCR was carried out using Hieff ® qPCR SYBR Green Master Mix (11202ES08, YEASEN, China). The qRT-PCR cycle parameters were set at 95 for 2 min, 95 for 10 s, and annealing temperature at 60 for 40 s, which comprised a total of 40 cycles. Then the 2 -CT method was used to calculate the relative induction ratios of treated samples compared with controls (Livak and Schmittgen, 2001). Three biological replications were used and three measurements were performed on each replicate.
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