Total RNA was extracted using TRIzol Reagent (Invitrogen, USA), and the concentration and quality of the RNA were measured with a NanoPhotometer spectrophotometer (Thermo, USA). A Hiscript II Q Select RT kit (Vazyme, CHN) was used for RNA reverse transcription. PCR was performed using the iTaqTM Universal SYBR Green Supermix (Vazyme, CHN). The expression levels of RNA were analyzed via the 2−ΔΔCt method. The sequences of primers used in this study are listed in Table S1 .
Itaq universal sybr green supermix
Manufactured by Vazyme
Sourced in China
The iTaq™ Universal SYBR® Green Supermix is a ready-to-use qPCR reaction mix that contains all the necessary components for real-time PCR amplification using SYBR® Green detection. It is designed to provide reliable and sensitive quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nanophotometer spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hiscript 2 q select rt kit, by Vazyme (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Mirna 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Dna gel extraction kit, by Sangon (1 mentions)
Hiscript 3 rt supermix, by Vazyme (1 mentions)
Venetoclax, by MedChemExpress (1 mentions)
Pcr product purification kit, by Sangon (1 mentions)
Dna primers, by Sangon (1 mentions)
T7 high yield transcription kit, by Vazyme (1 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (1 mentions)
Firefly luciferase reporter gene assay kit, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BestBio (1 mentions)
Cfx384 thermocycler, by Bio-Rad (1 mentions)
4 protocols using itaq universal sybr green supermix
1
Quantitative RNA Expression Analysis
2
Quantitative Gene Expression Analysis
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The Cell RNA Extraction Kit (Vazyme, CHN) was used to isolate total RNA, and quantification was performed at 260–280 nm using a NanoPhotometer spectrophotometer (Thermo, USA). cDNA was synthesized by reverse transcription of RNA using Hiscript II Q Select RT Kit (Vazyme, CHN). RT-qPCR was performed utilizing the iTaqTM Universal SYBR Green Supermix (Vazyme, CHN). The expression level of gene was normalized to the expression level of GAPDH mRNA, and compared by 2−ΔΔCT method. Primer sequences were listed in the Additional file 4 : Table S4.
3
Optimized CRISPR-dCas9-VPR System
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DNA primers were ordered from Sangon Biotechnology (Shanghai, China). Escherichiacoli strain DH5α was obtained from MiaolingBio (Wuhan, China). SpCas9 nuclease, Phanta Max Super-Fidelity DNA Polymerase, Hiscript III RT SuperMix for quantitative polymerase chain reaction (qPCR), T7 High Yield Transcription Kit, iTaq Universal SYBR Green Supermix and miRNA 1st Strand cDNA synthesis kits were purchased from Vazyme (Nanjing, China). Lipofectamine 3000 was purchased from ThermoFisher (USA). Plasmid DNA was extracted using the Plasmid mini-prep kit (Vazyme), and PCR amplicons were purified using the DNA gel extraction kit or the PCR product purification kit (Sangon Biotechnology). T4 ligase and restriction enzymes were purchased from New England Biolabs. Vectors of pJ23119, pU6, pCMV and pACYC plasmids were obtained from Addgene. The HEK293T-dCas9-VPR was prepared through lentivirus infections as described in our previous work (26 (link)). Trizol reagent was purchased from Ambion (USA). The firefly Luciferase Reporter Gene Assay Kit was purchased from Beyotime (China). The Annexin V-FITC/PI apoptosis detection kit was purchased from BestBio (China). Venetoclax was purchased from MedChemExpress (MCE). Other reagents were obtained from Sangon Biotechnology, unless otherwise indicated.
4
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen) following to the manufacturer’s protocol. Exactly 1 μg of total RNA was reverse transcribed into cDNA using Reverse Transcription Master Mix (Tiangen). qPCR reactions were performed in 384-well plates on a CFX-384 thermocycler (Bio-Rad) using iTaq Universal SYBR Green Supermix (Vazyme) as illustrated by the manufacturer’s instructions. Primer sequences for the experiment are listed in Supplementary Table 1 .
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