GSE66331 Real-time quantitative PCR analysis of human monocyte cells treated invitro with purified angiotensin type 1 receptor auto antibody

Contributors : Peter Abadir ; Neal FedarkoSeries Type : Expression profiling by RT-PCROrganism : Homo sapiensHuman Peripheral blood mononuclear cells were purified from older volunteers' blood and were cultured for 24 hrs in the presence or abscence of angiotensin type 1 receptor autoantibody (AT1RaAb 7ug/ml). In brief: PBMCs were isolated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation (86). PBMCs from older individuals cultured in six-well plates (0.5 x 106 cells/ml) were divided into two groups: control group and treatment group. The treatment group was incubated with purified AT1R-aAb (Eagle bioscience, Noshua, New Hampshire) for 24 hours. Supernatant was collected and cells harvested after a total culture period of 24 hours. RNA extraction, RT2 profiler assay and pathway analysis: Control and AT1R-aAb treated monocytes and lymphocytes from participants were prepared for RNA isolation. Total cellular RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, D üren, Germany). cDNA was synthesized at 37°C for 60 minutes and the reaction was stopped by heating the reaction mix to 94 °C. PCR cycling parameters are 10 minutes at 95°C and 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. All primers for the polymerase chain reaction (PCR) were purc hased from Operon Technologies, Inc. (Alameda, CA). We performed quantitative PCR with an Mx-3000P Real Time PCR System Instrument and SYBR Green florescent reagents (Stratagen...
Source: GEO: Gene Expression Omnibus - Category: Genetics & Stem Cells Tags: Expression profiling by RT-PCR Homo sapiens Source Type: research