After the cells were transfected for 48 h, the mRNA levels of CHD5 were analyzed by qPCR assay. Briefly, total RNA was extracted from the cells using the total RNA extraction kit (Bioflux, Tokyo, Japan). 1.5 µg of RNA was reverse transcribed into cDNA and the mRNA level was analyzed by UltraSYBR Master (CWBIO, Guangzhou, China). The qPCR primers for CHD5 were as follows: F-CCCCATGTCCAAAATGATGACC, and R-GTGACCGTCTCTACAGCCG. The data were normalized to 18 s and the relative gene expression was obtained using Prism 7 (GraphPad Software, lnc., La Jolla, CA, USA).
Total rna extraction kit
Manufactured by BioFlux
Sourced in China, Japan
The Total RNA Extraction Kit is a laboratory tool designed to isolate and purify total RNA from a variety of biological samples. It utilizes a guanidinium thiocyanate-phenol-chloroform extraction method to effectively separate RNA from DNA, proteins, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (3 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (2 mentions)
Sybr qpcr master mix, by Takara Bio (2 mentions)
Paris kit, by Thermo Fisher Scientific (2 mentions)
Icycler iq5 real time pcr system, by Bio-Rad (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Prism 7, by GraphPad (1 mentions)
Nebnext ultra rna library prep kit, by New England Biolabs (1 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Nanophotometer, by Implen (1 mentions)
Access rt pcr system, by Promega (1 mentions)
Sybr green master mix, by Toyobo (1 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
8 protocols using total rna extraction kit
1
Quantifying CHD5 mRNA Expression
2
Total RNA Extraction and qPCR Analysis
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Total RNA from HCC cells or tissues was extracted using total RNA extraction kit (BioFlux, Hangzhou, China) according to the manufacturer’s instructions. For nuclear/cytoplasmic separation assay, cytoplasmic and nuclear RNA were separately isolated using PARIS kit (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. The RNA was reversely transcribed to first-strand cDNA using PrimeScript RT reagent kit (Takara, Dalian, China). qPCR was performed with SYBR qPCR master mix (Takara), taking GAPDH as the internal reference. Reverse transcription and qPCR of miRNAs were conducted with All-in-One miRNA qRT-PCR detection kit (GeneCopoeia, Guangzhou, China), taking U6 RNA as the internal control. The primers were listed in Supplementary Table 1 .
3
RNA-seq Library Construction Protocol
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For RNA-seq library construction, total RNA was extracted from three biological replicates and three mocks at one time point by using the Total RNA Extraction Kit (BioFlux, Tokyo, Japan). The infected samples named VIV1, VIV2, VIV3, for VIV and VID1, VID2, and VID3 for VID were collected at 0, 12, and 36 hpi. The RNA quality and purity were checked on 1% agarose gels by using a NanoPhotometer® spectrophotometer (Implen GmbH, München, Germany).
Sequencing libraries were generated using 3 μg of RNA per sample as input material and the NEB Next® Ultra™ RNA Library Prep Kit (NEB, Ipswich, MA, USA) for Illumina sequencing (Illumina Inc., San Diego, CA, USA). Index codes were added to each sample to tag the sequences. The index-coded samples were clustered on a cBot Cluster Generation System by using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina), following the manufacturer’s instructions. After the clusters were generated, the libraries were sequenced by Novogene (Beijing, China) on an Illumina HiSeq 2000 platform to generate 100 bp paired-end reads.
Sequencing libraries were generated using 3 μg of RNA per sample as input material and the NEB Next® Ultra™ RNA Library Prep Kit (NEB, Ipswich, MA, USA) for Illumina sequencing (Illumina Inc., San Diego, CA, USA). Index codes were added to each sample to tag the sequences. The index-coded samples were clustered on a cBot Cluster Generation System by using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina), following the manufacturer’s instructions. After the clusters were generated, the libraries were sequenced by Novogene (Beijing, China) on an Illumina HiSeq 2000 platform to generate 100 bp paired-end reads.
4
Quantitative Real-Time PCR Protocol
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Total cellular RNA was extracted from cells at the exponential phase using the total RNA extraction kit as described by the manufacturer (BioFlux, Beijing, China). RNA preparations were treated with DNase I to eliminate residual DNA. The cDNA was synthesized using RevertAidTM First Strand cDNA synthesis kit (Fermentas, Shanghai, China). The qRT-PCR was performed using the QIAGEN OneStep RT-PCR Kit (TIANGEN, Beijing, China) on iCycler iQ5 real-time PCR system (Bio-Rad, Richmond, USA). The PCR reaction system and procedure was set following our previous reports5 (link). The transcriptional levels were normalized to the 16S rRNA from the same RNA samples. Each sample was analyzed in triplicate.
5
Quantifying DNA-PKcs Expression in Tumors
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The expression levels of the DNA-PKcs of the four groups were analyzed by qPCR assay. Total RNA of tumor and adjacent normal mucosa tissue specimens were extracted using total RNA extraction kit (BioFlux Corporation, Tokyo, Japan) according to the manufacturer's protocol. cDNA was synthesized using an Access RT-PCR system (Promega Corporation, Madison, WI, America). qPCR was performed using SYBR® Green ER qPCR SuperMix-UDG with ROX (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The following thermocycling conditions were used: 50°C for 2 min; 95°C for 2 min; 95°C for 15 sec; and 60°C for 30 sec for 40 cycles. The primer sequences used for qPCR were as follows: DNA-PKcs, forward 5′-AGCATCATGGTACACGCACT-3′ and reverse 5′-TCCATCAGGCAC1TrCACTFG-3′; and β-actin, forward 5′-CCTCGCCTTTGCCGATCC-3′ and reverse 5′-GGATCTTCATGAGGTAGTCAGTC-3′. The length of the PCR products of DNA-PKcs and β-actin were 383 and 626 bp, respectively. Relative mRNA levels were calculated based on 2−∆∆Ct method (26 (link)). All experiments were performed in triplicate.
6
Gene Expression Quantification by qRT-PCR
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Total cellular RNA was extracted from cells at the exponential phase with the total RNA extraction kit (BioFlux, Beijing, China) as described by the manufacturer. To eliminate residual DNA, RNA preparations were treated with DNase I. The cDNA was synthesized using RevertAid™ First Strand cDNA synthesis kit (Fermentas, Shanghai, China). The qRT-PCR was performed using the QIAGEN OneStep RT-PCR Kit (TIANGEN, Beijing, China) on an iCycler iQ5 real-time PCR system (Bio-Rad, Richmond, USA). RNA (1 μg) and 0.6 μmol L−1 of each primer (final concentration) were added to the RT-PCR mixture (50 μL), and the primers used for qRT-PCR are listed in Additional file 1 : Table S2. The PCR procedure was prepared following the instructions of the kit (TIANGEN, Beijing, China). The target gene transcriptional levels were normalized to the 16S rRNA from the same RNA samples. Each sample was analyzed in triplicate.
7
Quantitative Gene Expression Analysis in Muscle Tissue
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Firstly, the total RNA extraction kit (BioFlux, Beijing, China) was used to extract the total RNA from the muscle (6 replicates per group). All samples were diluted to achieve the same RNA concentration. RNA reverse transcription was carried out using the PrimeScript TMRT reagent kit (Takara, Tokyo, Japan). The primers used in this experiment are shown in Table 4 . The qRT-PCR was assayed by SYBR® Green Master Mix (Toyobo Co., Ltd., Osaka, Japan) with a CFX Connect Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), which was previously described by Zhang et al. [33 (link)]. Finally, statistical analysis of all data was carried out with the 2−ΔΔCT method.
8
RNA Extraction and qPCR Analysis
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Total RNA from HCC cells or tissues was extracted using the total RNA extraction kit (BioFlux, Hangzhou, China) according to the manufacturer's instructions. For nuclear/cytoplasmic separation assay, cytoplasmic and nuclear RNA were separately isolated using PARIS kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The RNA was reversely transcribed to rst-strand cDNA using PrimeScript RT reagent kit (Takara, Dalian, China). qPCR was performed with SYBR qPCR master mix (Takara), taking GAPDH as the internal reference. Reverse transcription and qPCR of miRNAs were conducted with All-in-One miRNA qRT-PCR detection kit (GeneCopoeia, Guangzhou, China), taking U6 RNA as the internal reference. The primers were listed in Supplementary Table 1.
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