Tissue samples were suspended in RNAlater ICE (Ambion) and stored at −20°C. mRNA was isolated from RNAlater ICE stabilized tissues or directly from in vitro cultures of monolayers or cell-laden hydrogels with TRIzol reagent (Ambion). Tissue samples were homogenized with a Polytron PT 2100. Assays were carried out on an ABI 7500 RT-PCR system with TaqMan Universal Master Mix II and validated PrimeTime primer probe sets (Integrated DNA Technologies). A first-strand cDNA synthesis kit (Fisher) was used to transcribe 5 μg RNA/20 μL. cDNA (100 ng) was used per RT-PCR reaction in triplicates. The Δ-Δ-CT method was used to comparatively assess mRNA quantity. All data are represented as a sample’s value normalized to GAPDH relative to source tissue–derived ADMSCs, source tissue, or endogenous BAT.
Taqman universal master mix 2
Manufactured by Integrated DNA Technologies
Sourced in United States
TaqMan Universal Master Mix II is a ready-to-use solution for real-time PCR amplification and detection. It contains all the necessary components for a quantitative PCR reaction, including Taq DNA polymerase, dNTPs, and optimized buffer.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rnalater ice, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Validated primetime primer probe sets, by Integrated DNA Technologies (1 mentions)
Direct zol rna miniprep, by Zymo Research (1 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using taqman universal master mix 2
1
Quantitative RT-PCR Analysis of mRNA
2
Quantitative mRNA Expression Analysis
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mRNA was extracted from tissues or in vitro cultures with TRIzol reagent (Invitrogen, Waltham, MA, USA) and purified using Direct-zol RNA miniprep (R2055, Zymo, Irvine, CA, USA) following manufacture instructions with DNase treatment. cDNA was synthesized using a Maxima First Strand cDNA synthesis kit (K1672, Thermo Scientific, Waltham, MA, USA), and 10–20 ng cDNA was used for qPCR on a QuantStudio 5 real-time PCR system with TaqMan Universal Master Mix II and validated PrimeTime primer-probe sets (Integrated DNA Technologies, Coralville, IA, USA). The ΔΔCt method was used to comparatively assess gene expression. Primer sequences are listed in Table A1 .
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