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

ABSTRACT: Human 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, Düren, 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 purchased from Operon Technologies, Inc. (Alameda, CA). We performed quantitative PCR with an Mx-3000P Real Time PCR System Instrument and SYBR Green florescent reagents (Stratagene, La Jolla, CA). QPCR to determine AT1R expression was performed using the TaqMan gene expression assay mix (Applied Biosystems) after reverse transcription of RNA with the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA). To avoid DNA contamination in RT-PCR assays, samples were extensively processed using DNase 1 digestion. Gene expression profiling for inflammation and autoimmunity target genes was performed using RT2 profiler PCR arrays (SuperArray, Frederick, MD) following the manufacturer's instructions. In brief, 1 μg RNA was reverse transcribed with the RT2 profiler PCR array first strand synthesis assay (SuperArray, Frederick, MD) followed by real-time PCR with RT2 real-time PCR master mix Sybr green (SuperArray, Frederick, MD). PCR data from the treated and untreated cells were analyzed by the QIAGEN SuperArray group and the results reported as differential gene expression and statistical significance, fold change and p-value respectively.

ORGANISM(S): Homo sapiens

PROVIDER: GSE66331 | GEO | 2018/02/25

REPOSITORIES: GEO