Validation of Sepsis Biomarkers by RT-qPCR

A selection of differentially regulated miRNAs and mRNAs from NGS was further investigated by RT-qPCR in a new cohort of septic patients sampled upon admission to the ICU (n = 40 for miRNAs; n = 39 for mRNAs) as well as healthy volunteers (n = 23). Prior to RT-qPCR, we used geNorm [21 (link)] and NormFinder [22 (link)] to predict the most stably expressed miRNAs and mRNAs based on the NGS data set and selected potential reference candidates. Reverse transcription of miRNAs was carried out using 10 ng of total RNA and the miRCURY LNA RT Kit (QIAGEN, Hildesheim, Germany). Real-time PCR was performed using 3 µl of diluted cDNA and QIAGEN’s miRCURY LNA SYBR Green PCR Kit. For analysis of mRNAs, 300 ng of total RNA were reverse transcribed using the QuantiTect RT Kit (Qiagen, Hildesheim, Germany). We then used the Sso Advanced Universal SYBR Supermix and PrimePCR Assays (Bio-Rad, Munich, Germany) to quantify mRNA expression in 10 ng cDNA. All reactions were carried out in a 10 µl total reaction volume on a Rotor-Gene Q thermal cycler (QIAGEN, Hildesheim, Germany). Expression of miRNAs was normalized with the geometric mean of miR-625-3p, miR-501-3p and miR-30d-5p, while mRNAs were normalized with the geometric mean of Ribophorin I, Transmembrane BAX inhibitor motif containing 6 (TMBIM6) and Ubiquitin C. Relative quantification was carried out using the ΔΔCq method [23 (link)].