Computational protocol: Lipid Regulators during Atherogenesis: Expression of LXR, PPAR, and SREBP mRNA in the Human Aorta

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Protocol publication

[…] RNA was isolated by a homogenization of the frozen samples taken from the vessel walls in TRIzol Reagent (Invitrogen; Carlsbad, CA, USA). The RNA was then treated with chloroform, precipitated with isopropanol, and washed with ethanol. The synthesis of cDNA was performed on 2–6 µg of total RNA using a Promega ImProm_II™ Reverse Transcription System kit (Promega Corporation; Madison, WI, USA). The synthesized cDNA was used as a template for quantitative real-time polymerase chain reaction (qRT-PCR) on a Rotor-Gene 3000 amplifier (Corbett Research; Sydney, Australia) with a kit of reagents including the intercalating dye SYBR Green I (Syntol; Moscow, Russia) as recommended by the manufacturer. The details of amplification were described earlier . The primers for the most part of mRNAs were chosen using the Beacon Designer 6.00 program (www.PremierBiosoft.com) and described previously . The primers for the second house-keeping gene, guanine nucleotide-binding protein, beta-peptide 2-like 1 (GNB2L1) were the same as in report of Ishii et al . To check for the absence of products amplified from genomic DNA, isolated RNA was used as a template. Amplified products were sequenced using an ABI PRISM® BigDye™ Terminator v.3.1 kit of reagents and an ABI PRISM 3100-Avant automated DNA sequencer to confirm the expected sequences available in relevant databases. No new DNA sequences were revealed in this study. The results were included only when the melting temperature and the electrophoretic mobility of the amplified products corresponded to the expected values. DNA sequencing was performed in the Center of Collective Use “Genome” at the Engelhardt Institute of Molecular Biology. The content of the particular mRNAs was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA content as an internal reference control and expressed as percent. [...] The results were analyzed with the Statistica 7.0 program. Independent samples were compared using the Mann-Whitney U-test. Correlations were evaluated using the Spearman rank test. Differences or correlations were considered significant at p<0.05. Mean ± SD values are shown. Correlation graph weight values Gh were calculated as sum of modules of statistically significant pairwise correlation coefficients (rkl): Gh = ∑ |rkl| . […]

Pipeline specifications

Software tools Beacon Designer, Statistica
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Atherosclerosis