Total RNA was obtained from the tissue samples, and cells were harvested using TRIzol kits (Invitrogen Life Technologies). Quantitative PCR was performed using an Applied Biosystems 7900 Real-time PCR System (Shanghai, China) and a TaqMan Universal PCR Master Mix (Takara, Dalian, China), according to the manufacturer’s instructions.
Taqman universal pcr master mix
Manufactured by Takara Bio
Sourced in China, Japan
TaqMan Universal PCR Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to facilitate efficient and accurate amplification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Cdna synthesis supermix, by Takara Bio (2 mentions)
Sybr qpcr supermix plus, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
7900 real time pcr system, by Takara Bio (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using taqman universal pcr master mix
1
Quantitative PCR for RNA Analysis
2
Quantitative Real-Time PCR (qRT-PCR) Protocol
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qRT-PCR was conducted as described previously.18 (link) The whole brain or PAs were homogenized and mixed with Trizol reagent for RNA extraction. The yields of RNA were assessed using a Nanodrop 2000 spectrophotometer. cDNA synthesis was conducted using a cDNA Synthesis Supermix (TAKARA, Japan). qRT-PCR assays were performed using the SYBR qPCR Supermix Plus (TAKARA, Japan). β-actin was used to normalize gene expression data. To detect miRNA expression, total RNA was reverse transcribed and then mixed with TaqMan Universal PCR Master Mix (TAKARA, Japan) and miRNA-specific TaqMan primers (Springen, Nanjing, China). MiRNA expression data were normalized to U6 RNA. The fold-change of gene expression was measured using the 2−ΔΔCt method.18 (link) All primer sequences for qRT-PCR are presented in Supplementary Table S1
3
RNA Extraction and qRT-PCR Analysis
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Whole brains or astrocytes were homogenized and dissolved in Trizol (Invitrogen, Carlsbad, CA, USA) to extract RNA. The concentrations of RNA were measured by a Nanodrop 2000 spectrophotometer (Thermo Fisher, USA). Reverse transcription was performed using a cDNA Synthesis Supermix (TAKARA, Kusatsu, Shiga, Japan). The cDNA was subjected to qRT-PCR with the SYBR qPCR Supermix Plus (TAKARA). Expression data were normalized to β-actin. In addition, the total RNA was reverse transcribed to determine the miRNA expression, and the resulting cDNA was mixed with miRNA-specific TaqMan primers (springen, Nanjing, China) and TaqMan Universal PCR Master Mix (TAKARA). U6 RNA was used as an endogenous control for data normalization. Relative changes in expression were measured using the comparative threshold cycle (Ct) method and 2-ΔΔCt as described,21 (link) and the results indicated the fold change of expression. These primers for qRT-PCR are shown in Supplementary Table S1
4
Quantification of Hepatic and Adipose RBP4 mRNA
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Total RNA was isolated from the liver and subcutaneous adipose tissue using TRIzol (Invitrogen, Carlbad, CA, USA), and two μg of total RNA were reverse-transcribed into cDNA (Takara, Tokyo, Japan). In each RT-PCR reaction, 2 μl of cDNA was amplified in a final volume of 20 μl PCR reaction mixture using the TaqMan universal PCR master mix (Takara, Tokyo, Japan). Samples were incubated in the Perkin-Elmer PCR System 9700 (Applied Biosystems, Foster, CA, USA) for an initial denaturation at 95 ºC for 10 min, followed by 35 PCR cycles, with each cycle consisting of 95 ºC for 30 s, 62 ºC for 30 s, and 72 ºC for 40 s. The following primers were used: rat RBP4 (accession no. XM_215285.4) 5′-GACAAGGCTCGTTTCTCTGG -3′ (sense) and 5′-AAAGGAGGCTACACCCCAGT -3′ (antisense), and rat β-actin (accession no. NM_007393.1) 5′-CACGATGGAGGGGCCGGACTCATC -3′ (sense) and 5′-TAAAGACCTCTATGCCAACACAGT -3′ (antisense). The specificity of the PCR was further verified by subjecting the amplified products to agarose gel electrophoresis based on the expected product size. The mRNA levels of RBP4 and PPARγ were normalized to the internal control β-actin.
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