Total intracellular RNA was isolated using the RaPure Total RNA Kit (Magen). To analyze the POWV RNA levels in replicon cells, quantitative RT-PCR was performed. In brief, 1 µg total RNA was reverse transcribed using the ReverTra Ace qPCR RT Kit (TOYOBO, FSQ-101) to produce cDNA with random primers. Reactions of qPCR were carried out using the 2× RealStar Green Power Mixture (Genstar, A311) according to the manufacturer’s instructions. The qPCR primers for viral RNA were as follows—POWV: THU-5193 (5′-CGC CCT CAA CAC CAT CAC AAA C-3′) and THU-5194 (5′-TCA ACT CCG TGC TCC TTC AAC C-3′). The sequences of the qPCR primers for GAPDH were described previously (45 (link)). Relative expression levels of the target genes were calculated using the comparative cycle threshold method. All data were normalized relative to GAPDH.
Rapure total rna kit
Manufactured by Magen Biotechnology Co
Sourced in China
The RaPure Total RNA Kit is a laboratory equipment designed for the isolation and purification of total RNA from a variety of biological samples. It utilizes a silica-based membrane technology to efficiently capture and purify RNA, ensuring high-quality results for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Hiscript 2 one step qrt pcr sybr green kit, by Vazyme (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Realstar green power mixture, by GenStar (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
All in one rt mastermix, by Applied Biological Materials (1 mentions)
Hiscript 3 rt supermix, by Vazyme (1 mentions)
Chamq sybr qpcr master mix, by Vazyme (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Lightcycler, by Roche (1 mentions)
Reverse transcriptase, by Promega (1 mentions)
Hiscript 2 q rt supermix for qpcr, by Vazyme (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Transcript all in one first strand cdna synthesis supermix for qpcr, by Transgene (1 mentions)
Chamq sybr qpcr master mix 2, by Vazyme (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Quantinova sybr green pcr kit, by Qiagen (1 mentions)
All in one rt supermix perfect for qpcr kit, by Vazyme (1 mentions)
14 protocols using rapure total rna kit
1
Quantification of POWV RNA Levels
2
Quantitative mRNA Analysis in Murine Macrophages
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For mRNA analysis, total mRNA was isolated from immortalized murine macrophage using RaPure Total RNA Kit (MAGEN, Cat. No. R4011-02), and cDNA was synthesized from 1 μg total RNA using 5X All-In-One RT Mastermix (Applied Biological Materials, Cat. No. G492). qPCR was performed using MonAmpChemoHS qPCR Mix (Monad, Cat. No. MQ00101) with the instrument CFX96 Touch Real-Time PCR Detection System (BioRAD). Primer pairs used in this study are listed in Supplementary Table 1 .
3
Transcriptome Sequencing of YAP-KD
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Transcriptome sequencing and analysis between the control and YAP-KD group was carried out with the assistance of Bioprofile, Shanghai. Total RNA was extracted with a standard protocol of RaPure Total RNA Kit (Magen, Guangzhou, Cat No. R4011-02). Q20, Q30, and GC content parameters were used to assess the data quality (Supplementary Table S1 ).
4
RNA Isolation and qPCR Analysis
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RNA was isolated by RaPure total RNA Kit (Magen) following the manufacturer’s instructions. One microgram of RNA was used for reverse transcription with gDNA wiper HiScript III RT Super Mix (Vazyme). qPCR was performed using cDNA as template for amplifying the indicated targets listed in S2 Table by using ChamQ SYBR qPCR Master Mix (Vazyme) according to the manufacturer’s instructions. Transcript levels of each gene were quantitatively assessed by comparative CT values and normalized with control groups. Fold changes were calculated using 2−ΔΔCT method and the error bars represent standard deviation of three experiment replicates.
5
Quantitative Analysis of Gene Expression
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The total RNA was extracted from HCC cells using RaPure Total RNA Kit (Magen, Guangzhou, China). Then, complementary DNA was synthesized using the PrimeScript RT reagent Kit (Takara, Kusatsu, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with SYBR Premix ExTaq (Takara) on a LightCycler (Roche, Indianapolis, IN). Conditions for reverse transcription polymerase chain reaction were 30 seconds at 95°C and followed by 40 cycles of 95°C for 5 seconds. The primers were as following: CTSC, forward: 5′-CCAACTGCACCTATCTTGACC-3′ and reverse: 5′-AAGGCAAACCACTTGTAGTCATT-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward: 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse: 5′-GCCATCACGCCACAGTTTC-3′; TNF-α, forward: 5′-GAGGCCAAGCCCTGGTATG-3′ and reverse: 5′-CGGGCCGATTGATCTCAGC-3′. GAPDH was used as an internal control for semi-quantitation. All qRT-PCR experiments were performed in triplicate.
6
Quantifying Total RNA in Liver Tissues
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Total RNA in liver tissues and cells was extracted using TRIzol reagent or RaPure total RNA kit (#R4011, Magen, Guangzhou, China) according to the manufacturer’s instructions. The quality and concentrations of total RNA were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrot Inc., Wilmington, DE, USA). RNA was quantified by one-step real-time quantitative reverse transcription PCR (qRT-PCR) using the HiScript II One Step qRT-PCR SYBR Green Kit (#Q221-01, Vazyme, Nanjing, China). The expression of GAPDH was used as an internal control. The primer sequences are listed in Supplementary Table S1 .
7
RNA Isolation and Plasmid Generation
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RaPure Total RNA kit (Magen) was used to isolate total RNA according to the manufacturer's instructions. cDNA was synthesized using reverse transcriptase (Promega). CUL4A, CUL4B, DDB1, MMS22L and ESCO2 plasmids for transient expression were generated previously (22 (link)). GSK3A and GSK3B full-length genes were generated by PCR and were inserted into the pRK5-FLAG vector. All genes were sub-cloned into other vectors when necessary. See Supplementary Table S2 for details of plasmids used in this study, Supplementary Table S3 for plasmids generated in this study, and Supplementary Table S4 for primers used in this study.
8
RNA Extraction and RT-qPCR Analysis
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RNA extraction was performed with RaPure Total RNA Kit (Magen, R4011). One microgram of total RNA was then reverse-transcribed with HiScript II Q RT SuperMix for qPCR (Vazyme, R123-01). The primers used are listed in table S2. All the experiments were repeated three times.
9
Quantitative RNA Expression Analysis
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Total RNA was isolated using RaPure Total RNA Kit (Magen, R4011-03). Then, RNA was used to perform the reverse transcription for each sample with primer for 1 h at 37 °C. The minus-Gluc-RT primer sequence is 5′-ACTGTCGTTGACAGGACACG-3′ and plus-Gluc-RT primer sequence is 5′-TGGATCTTGCTGGCGAATGT-3′. Real-time quantitative PCR was carried out with the SYBR Green Master Mix (Applied Biosystems, A25742) on an ABI StepOnePlusTM system. Levels of mRNA expression were calculated and levels of actin mRNA was used for normalization. The primers used in real-time PCR analysis were synthesized by Biomed Beijing and their sequences are presented in supplementary Table S2 .
10
Quantifying Inflammatory Gene Expression
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Total RNA was extracted by using the RaPure Total RNA kit (Magen), which was used to generate cDNA by using TranScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech). qPCR was performed using the Bio-Rad sequence system CFX96 with ChamQ™ SYBR® qPCR Master Mix (2×) (Vazyme) as recommended by the manufacturer. The primers used were as follows: IL-6F: 5′-TCCAGTTGCCTTCTCCC-3′, R: 5′-GCCTCTTTGCTGCTTTC-3′; MIF F: 5′-CGCAGAACCGCTCCTACA-3′, R: 5′-GAGTTGTTCCAGCCCACATT-3′.β-actin (F: 5′- CATCCGCAAAGACCTGTACG-3′, R: 5′- CCTGCTTGCTGATCCACATC-3′) was used as the internal control. qPCR results were analyzed and converted to fold changes.
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