Total RNA from liver and gastrocnemius muscle was extracted using the Tri-Reagent kit (Molecular Research Center, Cincinnati, OH, USA), and gene expression was assessed by quantitative reverse transcription-polymerase chain reaction (PCR) (ABI Universal PCR Master Mix; Thermo Fisher Scientific, Waltham, MA, USA) using a Stratagene Mx3000P thermocycler (Stratagene, La Jolla, CA, USA). The gene expression data were normalized to the house keeping gene 18s. The primer and probe sets used in the assays were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and are further described in Table 1 .
Abi universal pcr master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Universal PCR Master Mix is a premixed solution containing all the necessary components for performing polymerase chain reaction (PCR) experiments. It includes a heat-stable DNA polymerase, dNTPs, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
Tri reagent kit, by Molecular Research Center (8 mentions)
Applied biosystems quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Magnetic activated cell sorting system, by Miltenyi Biotec (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
7500 fast rt pcr machine, by Thermo Fisher Scientific (1 mentions)
9 protocols using abi universal pcr master mix
1
Quantifying Gene Expression in Liver and Muscle
2
RNA Extraction and Gene Expression Analysis
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Total RNA from adipose tissue was extracted using the Tri‐Reagent kit (Molecular Research Center, Cincinnati, OH), and gene expression was assessed by quantitative reverse transcription‐polymerase chain reaction (PCR) (ABI Universal PCR Master Mix; Thermo Fisher Scientific, Waltham, MA) using a Stratagene Mx3000P thermocycler (Stratagene, La Jolla, CA). The gene expression data were normalized to the housekeeping gene cyclophilin. The primer and probe sets used in the assays were purchased from Thermo Fisher Scientific (Waltham, MA).
3
Quantitative RT-PCR Analysis of Liver RNA
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Total RNA from liver was extracted using the Tri-Reagent kit (Molecular Research Center, Cincinnati, OH) and gene expression was assessed by quantitative reverse transcription- (RT-) PCR (ABI Universal PCR Master Mix, Applied Biosystems, Foster City, CA) using a Stratagene Mx3000p thermocycler (Stratagene, La Jolla, CA). Cyclophilin was used to normalize the gene expression data. The primer and probe sets used in the assays were purchased from Applied Biosystems/Life Technologies (Grand Island, NY).
4
Chemokine Expression in Aortic Tissue
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For chemokine expression in aortas, the whole aortas (from the sinotubular junction to the iliac bifurcation) were dissected and immediately frozen in liquid nitrogen. Total RNA was extracted using the TRI Reagent kit (Molecular Research Center, Cincinnati, OH), and gene expression was assessed by quantitative RT-PCR using ABI Universal PCR Master Mix (Applied Biosystems, Foster City, CA) using a Stratagene Mx3000p thermocycler (Stratagene, La Jolla, CA). Cyclophilin was used to normalize the gene expression data. The primer and probe sets used in the assays were purchased from Applied Biosystems/Life Technologies (Grand Island, NY).
For gene expression in aortic macrophages, the digested aortas were used to isolate macrophages with rat anti-mouse F4/80 antibodies (AbD Serotec), followed by a pull down of F4/80-positive cells with sheep anti-rat microbeads using a magnetic-activated cell sorting system (Miltenyi Biotec, Auburn, CA), as described previously (22 (link),23 (link)). The isolated F4/80-positive aortic macrophages were used for RNA extraction with TRI Reagent (Molecular Research Center) and gene expression analysis as described above (22 (link),23 (link)).
For gene expression in aortic macrophages, the digested aortas were used to isolate macrophages with rat anti-mouse F4/80 antibodies (AbD Serotec), followed by a pull down of F4/80-positive cells with sheep anti-rat microbeads using a magnetic-activated cell sorting system (Miltenyi Biotec, Auburn, CA), as described previously (22 (link),23 (link)). The isolated F4/80-positive aortic macrophages were used for RNA extraction with TRI Reagent (Molecular Research Center) and gene expression analysis as described above (22 (link),23 (link)).
5
Quantitative RT-PCR for Gene Expression
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Cellular RNA was extracted from cultures using the Tri Reagent kit (#RT 111, Molecular Research Center, Cincinnati, OH, USA), as described in the manufacturer’s protocol for cells grown in monolayer. ABI Universal PCR Master Mix and TaqMan primer and probe pair reagents were acquired from Applied Biosystems/ThermoFisher Scientific. Gene expression was evaluated using quantitative RT-PCR using Applied Biosystems QuantStudio 3 real-time PCR system (Applied Biosystems/ThermoFisher Scientific). Target mRNA expression levels were normalized to the expression of the housekeeping gene, cyclophilin, relative to controls. We used cyclophilin primer and probe sequences: 5′-GGTGGAGAGCACCAAGACAGA-3′(forward), 5′-GCCGGAGTCGACAATGATG-3′(reverse), and 5′-TCCTTCAGTGGCTTGTCCCGGCT-3′(probe). All other gene expression primers and probes were purchased from Applied Biosystems. Corresponding cycle threshold (Ct) values were measured, and relative mRNA level was determined using the 2(−ΔΔCt) method.
6
Quantitative RT-PCR Expression Analysis
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Total RNA of the 3T3-L1 cells and ST2 cells was extracted using the Tri Reagent kit (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s protocol. The expression of genes of interest was assessed by quantitative RT-PCR (ABI Universal PCR Master Mix, Applied Biosystems, Foster City, CA) using a Stratagene Mx3005p thermocycler (Stratagene, La Jolla, CA), as we previously described40 (link)41 (link). The primer and probe pairs used in the assays were purchased from Applied Biosystems (Foster City, CA).
7
Quantitative Real-Time RT-PCR for mRNA Expression
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Total RNA was extracted from BAT1/HIB-1B cells using Tri-Reagent kit (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instruction. The quantitation of the mRNAs of genes of interest was assessed by quantitative real-time RT-PCR (ABI Universal PCR Master Mix, Applied Biosystems, Foster City, CA) using a Stratagene Mx3005p thermocycler (Stratagene, La Jolla, CA) and was normalized by the corresponding cyclophilin mRNA measurement as we previously described46 (link),47 (link). Some primer and probe pairs used in the assays were purchased from Applied Biosystems and the primer/probe sequences for other genes are provided in Summplemental Table 1 .
8
Quantitative RT-PCR Analysis of Liver Gene Expression
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Total RNA was extracted from liver using the Tri‐Reagent kit (Molecular Research Center, Cincinnati, OH) as it was previously described.[42 ] The expression of genes of interest was quantitated by quantitative RT‐PCR (ABI Universal PCR Master Mix, Applied Biosystems, Foster City, CA) using an Applied Biosystems QuantStudio 3 real‐time PCR system (ThermoFisher Scientific).[42 ] The primer and probe pairs used in the assays were purchased from Applied Biosystems.
9
RNA Extraction and qRT-PCR Analysis
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RNA from frozen tissues was extracted using TRIzol Reagent (Thermo Fisher) according to the manufacturer’s instructions. Gene expression was measured using a 7500-Fast RT-PCR machine (Applied Biosystems), ABI Universal PCR Master Mix (Applied Biosystems, Foster City, CA), and primer and probe sets purchased from Applied BioSystems. Relative gene expression was determined using the ΔΔCt method with cyclophilin as an internal control (88 (link)).
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