RNA Extraction and Real-Time PCR Analysis

Total mRNA was extracted from 1 × 10⁶ cells by Manual PerfectPure^TM RNA Cell & Tissue (5PRIME, GmbH). RNA was reverse transcribed using the high-capacity cDNA Archive Kit (Applied Biosystems, Foster City, USA) and real time PCR were performed as previously described [21 (link)]. Finally, raw data were analyzed by the ABI prism SDS software (Applied Biosystems) and primers (Ct1) mRNA expression data were obtained from Ct values, normalized to 18 s RNA (Ct2) (housekeeping) content and finally expressed as 2^-Δct for each gene.

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Guasti L., Maresca A.M., Schembri L., Rasini E., Dentali F., Squizzato A., Klersy C., Robustelli Test L., Mongiardi C., Campiotti L., Ageno W., Grandi A.M., Cosentino M, & Marino F. (2016). Relationship between regulatory T cells subsets and lipid profile in dyslipidemic patients: a longitudinal study during atorvastatin treatment. BMC Cardiovascular Disorders, 16, 26.

Publication 2016

high-capacity cDNA Archive Kit ABI prism SDS software

Cdna Cell Gene Mrna Primers Prism Real time pcr Tissue

Corresponding Organization :

Other organizations : University of Insubria, University of Pavia

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Variable analysis

independent variables

Total mRNA extracted from 1 × 10^6 cells

dependent variables

MRNA expression data (2^-ΔCt) for each gene

control variables

18s RNA (Ct2) as a housekeeping gene

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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