Illumina HiSeq 2500 sequencing; MicroRNA profiling from pooled plasma samples of patients with sepsis and systemic inflammatory response syndrome (SIRS)
MicroRNA profiling from pooled plasma samples of patients with sepsis and systemic inflammatory response syndrome (SIRS)
Plasma was fractionated from blood and cryopreserved (-80°C). To control for the bias of miRNAs from red blood cells in differential analysis, we compiled pools so that average levels of hemolysis were comparable across groups. After exclusion of hemolysis specimens, plasma pools were formed by combining equal volumes of patients’ plasma. Total RNA was extracted from 2.5 ml plasma using the miRVana PARIS technology kit (Life Technologies). NGS cDNA libraries were prepared and validated from plasma RNA by ARK Genomics (University of Edinburgh, UK), following manufacturer instructions, with specific barcodes for each cDNA library (Illumina TruSeq Small RNA sample protocol). Bands corresponding to ~22 nucleotides in the original sample were size-selected (correct insert size: 146bp) after band staining and visualization under UV-light.