Title: Affymetrix CEL analysis. Description:
RNA was isolated using the RNeasy Plant Mini Protocol (QIAGEN, Clifton Hill, Australia) and the quality of the RNA was assessed using the Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) where spectrophotometric analysis was carried out to determine the A260 to A280 ratio and the concentration. For qRT-PCR, cDNA synthesis was carried out using the Biorad (Perth) iScript cDNA synthesis kit according to manufacturers instructions. For arrays, the cRNA was prepared from 3 ug of total RNA almost exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Title: GeneChip hybridization. Description:
nucleic acid extraction protocol
Qiagen Paxgene Blood RNA manual protocol, Ambion WT Expression kit (Parameters: Extracted product = total_RNA, Amplification = none)
Patients who presented to Nottingham University Hospital Pediatric Intensive Care Unit (PICU) were recruited, and parents gave full written consent on behalf of children who took part in the study. Blood samples were obtained on admission (0 hours), and at 4, 8, 12, 24, and 48 hours during their PICU admission. Blood was acquired by nursing staff from the patients' central lines. One ml of blood was incubated with 3ml PAXgene Blood RNA Tube additive (PreAnalytiX PAXgene Blood RNA Tube, Qiagen/BD) at room temperature for 30 minutes to 1 hour, and then transferred to -70 degrees celsius. To extract RNA, the blood mixture samples were thawed, further incubated at room temperature for 1 hour, and extraction was carried out as per Qiagen PAXgene Blood RNA Kit protocol.