Total RNA was extracted from epididymal adipose tissue using the reagent TRIzol (Invitrogen). The SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen) kit was used for the synthesis of 20 μl of complementary DNA from 1000 ng of total RNA. The mRNA levels of PPAR-γ (Rn00440945_m1, Applied Biosystems) was determined by real-time PCR. Quantitative measurements were made with a commercial kit (TaqMan qPCR; Applied Biosystems) in a detection system (StepOne Plus; Applied Biosystems). Cycling conditions were as follows: enzyme activation at 50 °C for 2 min, denaturation at 95 °C for 10 min, complementary DNA products were amplified for forty cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Gene expression was quantified in relation to the values of the C group after normalization by an internal control (cyclophilin: assay Rn 00690933_m1; Applied Biosystems) by the method 2-ΔΔC T, as described previously (Livak and Schmittgen, 2001 (link)).
Rn00440945 m1
Manufactured by Thermo Fisher Scientific
The Rn00440945_m1 is a laboratory instrument designed for use in scientific research and analysis. It is a key piece of equipment used in various applications within the life sciences and scientific fields. The core function of this product is to perform specific analytical tasks, though the exact details of its intended use cannot be provided in an unbiased and factual manner within the constraints of this request.
Automatically generated - may contain errors
Lab products found in correlation
Taqman qpcr, by Thermo Fisher Scientific (1 mentions)
Superscript 2 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Cyclophilin assay rn 00690933 m1, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Prism 7000, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay kit, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Isogen 2, by Nippon Gene (1 mentions)
Taqman pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rn00562126 m1, by Thermo Fisher Scientific (1 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (1 mentions)
3 protocols using rn00440945 m1
1
Quantifying PPAR-γ Expression in Adipose Tissue
2
Quantitative RT-PCR Analysis of Rat PT Cells
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Total RNA was extracted from isolated rat PTs with isogen II (Nippon Gene), according to the manufacturer's instructions, and first-strand complementary DNA was synthesized using a cDNA Synthesis Kit (Takara), as previously reported (20 (link)). The mRNA expression levels were estimated using quantitative PCR (Prism 7000; Applied Biosystems) with TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan Gene Expression Assay kits, Rn00440945_m1 for rat PPARγ, Rn00580728_m1 for rat CD36, or Rn00667869_m1 for rat β-actin (Applied Biosystems). The mRNA levels were normalized to β-actin expression levels.
3
Quantitative RT-PCR Analysis of BAT
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Total RNA was extracted from the hypothalamus, pituitary, and interscapular BAT according to the Tri-Reagent protocol [50 (link)]. The reverse transcription reaction was carried out on 2 μg of RNA using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using TaqMan PCR Master Mix and the thermocycler parameters recommended by the manufacturer. PCRs were performed in a volume of 50 μL, containing 25 μL of the reverse transcription reagents. TaqMan gene expression assays were used for ACCα, CPT1b, FASN, GH, GLUT4, IGF-IR, PPARγ, SRIF, sst2, and UCP-1 (Rn00573474_m1, Rn00682395_m1, Rn00569117_m1, Rn01495894_g1, Rn00562597_m1, Rn01477918_m1, Rn00440945_m1, Rn00561967_m1, Rn00571116_m1 and Rn00562126_m1, respectively; Applied Biosystems). Relative gene expression comparisons were carried out using an invariant endogenous control (actin, Rn00667869_m1). The ΔΔCT method was used for relative quantification.
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