The total RNA was extracted by using Trizol (Ambion, United States). The High Capacity cDNA Reverse Transcription Kit (Vazyme, Nanjing, China) was used to conduct the reverse transcription. SYBR Green chemistry (Vazyme, Nanjing, China) on a 7,500 fast RT-PCR system was used to perform amplified reaction. The primers (Invitrogen Co, Shanghai, China) were listed in Supplementary Table S1 . The ratio of the mRNA expression of the target gene vs that of β-actin was defined as 2−△△Ct.
High capacity cdna reverse transcription kit
Manufactured by Vazyme
Sourced in China
The High-Capacity cDNA Reverse Transcription Kit is a reagent kit designed for the reverse transcription of RNA to cDNA. The kit contains the necessary components to efficiently convert RNA into first-strand cDNA for downstream applications such as PCR or qPCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Sybr green chemistry, by Vazyme (2 mentions)
Trizol, by Vazyme (2 mentions)
Sybr green master mix, by Vazyme (2 mentions)
Sybr green pcr master mix, by Vazyme (2 mentions)
Sybr green real time pcr kit, by Vazyme (1 mentions)
Minioption thermocycler, by Bio-Rad (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
Sybr qpcr master mix, by Vazyme (1 mentions)
Sybr green kit, by Qiagen (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Sybr green qpcr kit, by Vazyme (1 mentions)
Lightcycler 480 pcr system, by Roche (1 mentions)
Lightcycler 480 2 real time pcr instrument, by Roche (1 mentions)
Quantstudio 5, by Vazyme (1 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Abi 7900 pcr system, by Thermo Fisher Scientific (1 mentions)
19 protocols using high capacity cdna reverse transcription kit
1
Quantitative Real-Time PCR Analysis
2
RNA Extraction and qRT-PCR Analysis
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The total RNA was extracted by using Trizol (Ambion, USA). The High Capacity cDNA Reverse Transcription Kit (Vazyme, Nanjing, China) was used to conduct the reverse transcription. Thermo Fisher Scientific synthesized the primers, and the primer sequences are summarized in the Table S6 . SYBR Green chemistry (Vazyme, Nanjing, China) on a 7500 fast RT-PCR system was used to perform an amplified reaction. The amount of target, normalized to an endogenous reference (β-actin), was given by the 2−ΔΔCT calculation.
3
Gene Expression Analysis in Rat Model
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Rats in both groups (n = 6) were deeply anesthetized with sodium pentobarbital (200 mg/kg, i.p.) on PODs 7, 14, 21, and 28. Total RNA was extracted in TRIzol reagent (Invitrogen, TX, United States, Cat# 15596018). Subsequently, a High-Capacity cDNA Reverse Transcription Kit (Vazyme, Nanjing, China, Cat# R223) was used to convert RNA into cDNA, while a SYBR-Green Real-Time PCR Kit (Vazyme, Cat# Q311) and a Bio-Rad MiniOption thermocycler (Bio-Rad, CA, United States) were used for detection. Primer sequences are provided in Table 1 . The relative expression levels of RNA were evaluated using the 2−ΔΔCt method.
4
Transcriptome Analysis of Spermatogenic Cells
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Total RNA was extracted from testes and isolated spermatogenic cells using TRIzol reagent. Total RNA (1 μg) was reverse transcribed using a high-capacity cDNA reverse transcription kit (Vazyme) according to the manufacturer’s instructions. 2 μl diluted cDNA was subjected to real-time PCR using the SYBR Q-PCR master mix (Vazyme). The primers are listed in Table S8 . The relative gene expression was quantified using the comparative cycle threshold method, with the Arbp expression used for normalization. For RNA-seq, 1 μg total RNA extracted from testes or isolated spermatogenic cells was used to prepare the mRNA libraries using TruSeq Stranded mRNA Library Preparation Kit Set A (Cat. No. RS-122-2101; Illumina) according to the manufacturers’ instructions. All libraries were sequenced using the Illumina HiSeq 4000 platform. The FASTX-Toolkit was used to remove adaptor sequences, and low-quality reads from the sequencing data. Then, we used Tophat2 and Cufflinks to assemble the sequencing reads based on the UCSC MM10 mouse genome. The differential expression analysis was performed by Cuffdiff. The differential expressed genes were set with the threshold of FDR < 0.1 and fold change ≥ 2.
5
Quantitative Analysis of FOXM1 Expression
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Total RNA was extracted from tissues or cells using TRIzol reagent according to the instructions of the manufacturer (Invitrogen). The RNA purity was assessed by spectrophotometry (absorbance at 260 nm A260/A280 > 1.8). Approximately 1000 ng of total isolated RNA was subsequently transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Vazyme, China) according to the manufacturer’s instructions. qPCR was then performed for 1 min at 95 °C using a SYBR Green kit (Qiagen, Germany) on a Roche LightCycler 480 system with 200 nM primers, followed by amplification in 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 20 s. Relative expression was normalized to β-actin using quantification as 2−ΔΔCT (comparative threshold cycle). The primer sequences used were as follows: β-actin (forward: 5′-CACCATTGGCAATGAGCGGTTC-3′; reverse: 5′-AGGTCTTTGCGGATGTCCACGT-3′) and FOXM1 (forward: 5′-GGCCATCCCCAACAATGCTA-3′; reverse: 5′-AGGTCTCCAGGGTCACTTCT-3′).
6
Quantitative Gene Expression Analysis
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Total RNA was extracted from WAT and adipocytes using the RNA-quick Purification Kit (ES Science) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Vazyme Biotech Co., Ltd.) in accordance with the manufacturer's instructions. qPCR was performed using a SYBR Green QPCR kit (Vazyme Biotech Co., Ltd.) on a LightCycler 480 PCR System (Roche Diagnostics). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 2 min, followed by 40 cycles of 95˚C for 20 sec, 60˚C for 20 sec and 72˚C for 20 sec, and then a final step at 78˚C for 5 min. The fold changes in the expression levels of each gene were calculated using the 2-ΔΔCq method (27 (link)). The mRNA expression levels for each target gene were normalized to those of β-actin. The primer sequences are presented in Table II .
7
Validation of Transcriptome Analysis via qPCR
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Quantitative reverse transcription PCR (qPCR) was conducted to verify the transcriptome results. The exact procedure was described in the previous study (4 (link)). In brief, total RNA was extracted from homogenized colonic tissues in TRIzol® (Vazyme Biotech Co., Ltd., Nanjing, China). One-microgram purified RNA of each sample was firstly reverse-transcripted to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Vazyme Biotech Co., Ltd., Nanjing, China). qPCR was performed using LightCycler® 480 II Real-time PCR Instrument (Roche, Swiss). All samples were repeated for three times, and the relative expression of related genes was normalized relative to the endogenous reference (β-actin) with the 2-ΔΔCt method. Primer sequences were listed in Table 1
8
RNA Extraction and Differential Gene Expression Analysis
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Total RNA was extracted from cell tissues using a TRIzol reagent kit (Invitrogen). PCR was amplified and sequenced using Illumina NovaSeq 6000 by Gene Denovo Biotechnology (Guangzhou, China). We identified genes as significantly differentially expressed if they had a fold change ≥1 and a false discovery rate <0.05. Total RNA was isolated from osteosarcoma cell lines using the Total RNA Kit (R6834-01, Omega). One microgram of RNA was used for cDNA synthesis using a High-Capacity cDNA Reverse Transcription Kit (Q711-02, Vazyme). Transcript levels were determined on an Applied Biosystems QuantStudio 5 real-time PCR system using the Vazyme iTaq Universal SYBR Green Supermix (R223-01, Vazyme). Relative gene expression was calculated using the 2 -∆∆Ct method. The primer sequences are listed in Supplementary Table 2 .
9
Quantifying HIV-1 Gag Expression in Keap1-Silenced Cells
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293T cells were pre-treated with Keap1 target-specific siRNA or control siRNA and then infected with EIAV pseudotyped virus. Cells were collected at 12 h and 24 h post infection. To quantify the mRNA in siKeap1 and siCtrl cells, total RNA from the cells was extracted using a Bio-fast simply RNA extraction kit (catalog # BSC60S1, Bioer). Nuclear and cytoplasmic RNA fractions were isolated and purified using the PARIS Kit (catalog # AM1921, Thermo Scientific) according to the manufacturer’s instructions. An equal volume of RNA was used for cDNA synthesis using the High-Capacity cDNA reverse transcription kit (catalog # R223-01, Vazyme Biotech). Real-time PCR was then performed using the SYBR green PCR mixture with specific primers targeting gag. A relative quantification method was used, with Lamin and Tubulin protein as the nuclear and cytoplasmic protein controls, respectively. The following primers were used: Gag sense, 5’-GGGATTATTTGGTAAAGGG-3’, Gag anti-sense, 5’-GATTCTGCCATGCTGTTCT-3’.
10
Gene Expression Analysis via RT-PCR
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Total RNA was isolated using TRIzol (Life Technologies, USA). Then, a high-capacity cDNA reverse transcription kit (Vazyme, China) was used to reverse-transcribe 1 μg of mRNA into cDNA in accordance with the manufacturer’s protocol. RT-PCRs were performed on an ABI7900 PCR system (Applied Biosystems, USA) using SYBR Green MasterMix (Vazyme, China). GAPDH was used as a reference gene. The primers are shown in Supplementary Table 1 .
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