Blood samples were harvested directly and sequentially into 8 PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, CH) via a 21 − gauge butterfly needle and then frozen and kept at −80 °C. Total RNA was purified using PAXgene™ Blood RNA kit (PreAnalytiX GmbH, Qiagen, Hilden, Germany) and DNaseI treatment was performed by ‘on-column’ treatment as recommended by manufacturer’s instructions plus a second treatment subsequent to elution. RNA was then purified using RNeasy (Qiagen, Hilden, Germany) and quantified by NanoDrop ND-100 Spectrophotometer (NanoDropTechnologies; Wilmington, DE). RNA integrity was determined with 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and exclusively samples with RIN ≥ 8 were included in the subsequent investigations. Hybridization targets were synthesized with Ovation™ Whole Blood Solution (NuGEN) after comparison with other 2 methods (Additional file 1 ) and hybridized to HG − U133A 2.0 arrays (Affymetrix, Santa Clara, CA), investigating the expression of 18400 transcripts.
Ovation whole blood solution
Manufactured by Tecan
The Ovation™ Whole Blood Solution is a laboratory equipment product from Tecan. It is designed to extract and prepare whole blood samples for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hg u133a 2.0 arrays, by Thermo Fisher Scientific (1 mentions)
Paxgene blood rna tube, by Preanalytix (1 mentions)
Paxgene blood rna kit, by Preanalytix (1 mentions)
Nanodrop nd 100 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Encore biotin module, by Tecan (1 mentions)
Ez pcr core reagents, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
2 protocols using ovation whole blood solution
1
Whole Blood RNA Extraction and Profiling
2
RNA Extraction and Gene Expression Analysis
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RNA was extracted for RT-PCR and gene arrays with EZ-PCR Core Reagents (Life Technologies, Grand Island, NY), and custom primers were generated (see Table E2 in this article's Online Repository at www.jacionline. org for primers and probes). 16, 24, [34] [35] [36] [37] [38] Expression values were normalized to human acidic ribosomal protein (hARP). Human HGU133Plus2.0 GeneChip probe arrays (Affymetrix, Santa Clara, Calif) were used for gene arrays. 16, 24, [34] [35] [36] [37] [38] Total RNA was extracted with the Qiagen miRNeasy Mini Kit (Qiagen, Valencia, Calif), and DNA was removed with the Qiagen RNAse-free DNAse Set. Total RNA (50 ng) was reverse transcribed and amplified with Ovation Whole Blood Solution from NuGen (San Carlos, Calif). The labeled target was fragmented and hybridized to probe arrays by using the Encore Biotin Module from NuGen. For further details, see the Methods section in this article's Online Repository.
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