Title: Affymetrix CEL analysis. Description:
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
nucleic acid hybridization to array protocol
P-AFFY-3 GeneChip hybridization
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.