Quantitative RT-PCR Analysis of Aortic Gene Expression

Suprarenal aortas and perivascular and white adipose tissue were homogenized with Tissue Lyser using safe-lock tubes with metal bead and trizol-chloroform. Total aortic RNA was isolated with RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse transcribed with random primers and Superscript III (Invitrogen, Carlsbad, CA, USA). cDNA (1.0 ng) was amplified by RT-PCR with TaqMan Universal PCR Mastermix (Applied Biosystems, Foster City, CA, USA) using 7500 Fast Real-time PCR Sequence Detector (Applied Biosystems)11 (link)51 (link). Samples were run in duplicate and semi-quantified against a standard curve. All probes were obtained from Applied Biosystems and results were normalized to mouse TBP.

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Wågsäter D., Vorkapic E., van Stijn C.M., Kim J., Lusis A.J., Eriksson P, & Tangirala R.K. (2016). Elevated Adiponectin Levels Suppress Perivascular and Aortic Inflammation and Prevent AngII-induced Advanced Abdominal Aortic Aneurysms. Scientific Reports, 6, 31414.

Publication 2016

RNeasy Mini kit Superscript III TaqMan Universal PCR Mastermix 7500 Fast Real-time PCR Sequence Detector

Aortic Cdna Chloroform Metal Mouse Primers Real time pcr Rt pcr Tissue Trizol White adipose tissue

Corresponding Organization :

Other organizations : Linköping University, University of California, Los Angeles, Karolinska Institutet

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Variable analysis

independent variables

Tissue homogenization using Tissue Lyser with safe-lock tubes, metal bead, and Trizol-chloroform

dependent variables

Aortic RNA expression measured by RT-PCR using TaqMan Universal PCR Mastermix and 7500 Fast Real-time PCR Sequence Detector

control variables

Mouse TBP used for normalization of RT-PCR results

controls

Positive control: Standard curve used for semi-quantification of samples
Negative control: Not explicitly mentioned

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