Total mRNA was isolated from liver or muscle using Trizol (Thermo Fisher) and adipose tissues using Qiazol (Qiagen) according to the manufacturer's protocol. mRNA was converted to cDNA (Applied Biosystems # 4368814). Quantitative RT-PCR was performed on the liver and adipose tissue samples using the Bimake 2x SYBR Green qPCR Master Mix (catalog #B21203), with primers for target genes listed in Supplemental Table 1 . TBP, GAPDH, and Rpl39 mRNA levels were measured in each group and analyzed to determine the gene which best represented the total mRNA level. TBP was used for normalization unless otherwise specified.
Bimake 2x sybr green qpcr master mix
Manufactured by Selleck Chemicals
Sourced in United States
The Bimake 2x SYBR Green qPCR Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains SYBR Green I dye, hot-start DNA polymerase, dNTPs, and necessary buffers and reagents for efficient amplification and detection of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Qiazol, by Qiagen (1 mentions)
All in one cdna synthesis supermix, by Selleck Chemicals (1 mentions)
Fastpure cell tissue total rna isolation kit v2, by Vazyme (1 mentions)
All in one mirna first strand cdna synthesis kit, by GeneCopoeia (1 mentions)
Mircury rna isolation kit tissue, by Qiagen (1 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
3 protocols using bimake 2x sybr green qpcr master mix
1
Quantitative RT-PCR Analysis of Tissue mRNA
2
RNA Extraction and qPCR Analysis
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Seven NPC samples and four control samples were collected. All patients signed informed consent forms, which were approved by the hospital ethics committee. The diagnosis of all patients was confirmed by pathology. Total RNA from specimens was isolated using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, Nanjing, China) according to the manufacturer's instructions. mRNA to cDNA was synthesized using Bimake Allin-One cDNA Synthesis SuperMix (Bimake, Houston, USA), and qPCR of mRNAs was performed with Bimake 2x SYBR Green qPCR master mix (Bimake). Relative expression was calculated with the 2-DDCT method, and levels were normalized using GAPDH for mRNAs. The sequences of primers used in this study are shown in Table 3.
3
Comprehensive miRNA and mRNA Expression Analysis
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The total RNA for the specimens was isolated using miRCURY™ RNA Isolation Kit–Tissue (Exiqon, Vedbaek, Denmark), and the total RNA from cells was extracted using RNAiso Plus (Takara Biotechnology, Dalian, China) according to the manufacturer’s instructions. MiRNAs were reverse transcribed to cDNA using All-in-One™ miRNA First-Strand cDNA Synthesis Kit (GeneCopoeia, Guangzhou, China). The real-time quantitative PCR (qPCR) of miRNAs was performed with All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia). We synthesized mRNAs to cDNA using Bimake™ All-in-One cDNA Synthesis SuperMix (Bimake, Houston, USA), and the qPCR of mRNAs was performed with Bimake™ 2x SYBR Green qPCR master mix (Bimake). Relative expression was calculated with the 2−ΔΔCT method and levels were normalized using U6 and GAPDH for miRNAs and mRNAs, respectively. The primer sequences used in this study are shown in Table 2
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