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Real-time quantitative analysis of genes related to human innate & adaptive immune responses in neuroendocrine liver metastasis.

ABSTRACT: The immunological profile of patient-derived Precision Cut Tissue Slices (PCTS) obtained from neuroendocrine liver metastasis (LM-NEN=liver metastasis of neuroendocrine neoplasm) (n=3), cholangio carcinoma (CCA) (n=1) and tumour-free liver tissue (n=4) was analysed and the expression of 84 genes involved in innate and adaptive immune response was assessed. PCTS were kept in culture at 37C for 2 hours (recovery step) in a sealed chamber filled with carbogen under constant shaking prior to collection. Upon collection tissue slices were preserved in AllProtect Tissue Reagent (Qiagen) and stored at -80C until processing. RNA was isolated with Allprep Mini Kit (Qiagen) according to manifacturer's instructions. cDNA was synthesized with RT2 First Strand Kit (Qiagen) and analysed with Innate and Adaptive Immune Responses RT2 Profiler PCR Array (Qiagen) with RT2 SYBR® Green qPCR Mastermix (Qiagen). qPCR array for Human Innate & Adaptive Immune Responses (Qiagen 330231 PAHS-052ZA, full list of genes available online) was run on ABI 7500 Real-Time PCR System with an initial denaturation step at 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 60 seconds. More information about the histology of the slices used in this analysis can be found in the original manuscipt Doornebal et al.

ORGANISM(S): Homo sapiens

PROVIDER: GSE206828 | GEO | 2022/06/28

REPOSITORIES: GEO

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Project description: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.

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Real-time quantitative analysis of genes related to human innate & adaptive immune responses in neuroendocrine liver metastasis.

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