Primepcr assay

Total RNA was isolated from large intestine mucosal scrapes using a RNeasy kit (Qiagen, Valencia, CA, USA) and first-strand cDNA was generated using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). PrimePCR Assays (Biorad, Irvine, CA, USA) were used to perform quantitative RT-PCR including primers for CB₁R (Cnr1), Tight junction protein-1 (Tjp-1), Occludin (Ocln), and Claudin-1 (Cldn-1) gene transcripts under preconfigured SYBR Green assays (Biorad, Irvine, CA, USA). Identification of differential expression between intCB₁R−/− and intCB₁R+/+ control mice was performed using the common delta-delta (2^−ΔΔCq) method with Hprt as the housekeeping gene [47 (link)].

RNA was extracted from peripheral blood samples of 176 axSpA patients and 27 controls using the NucleoSpin RNA Blood Kit (Macherey-Nagel, Germany), according to the manufacturer’s instructions. Total RNA was reverse transcribed into complementary DNA (cDNA) using iScript^TM Advanced cDNA Synthesis Kit for reverse transcription- quantitative real-time PCR (qPCR) (Bio-Rad, USA), and cDNA was amplified with SsoAdvanced^TM Universal SYBR® Green Supermix (Bio-Rad, USA). qPCR reactions for omentin (target gene) and GAPDH (housekeeping gene) were performed in custom 384-well-plates (PrimePCR Assays, Bio-Rad, USA) in a 7900 HT real-time instrument (Applied Biosystems, USA). All samples were assayed in duplicate and experimental control assays were included. The relative omentin mRNA expression was analyzed by the comparative Ct method, as previously reported^{21 (link)}. Normalized values were obtained for each sample and mean values were determined for each study group.

Total RNA was extracted with the SV total RNA isolation system from Promega (Madison WI, USA) according to the manufacturer’s protocol. RNA quantity and purity were measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc. Wilmington DE, USA). RNA was reverse transcribed with the GoScript RT System (Promega) and qPCR was performed on a LightCycler480 thermal cycler (Roche Diagnostics, Basel Switzerland) using GoTaq qPCR master mix (Promega). Primers for CCL9, CXCL1 and CXCL2 were purchased as pre-tested and optimized PrimePCR Assays from Bio Rad (Hercules CA, USA). Primers for HPRT (fwd-5’ TCC TCC TCA GAC CGC TTT T 3’; rws-5’ CCT GGT TCA TCA TCG CTA ATC 3’) were designed using Primer3 software and purchased from Eurofins MWG Operon (Ebersberg, Germany). Specificity and efficiency was tested in initial analyses. Differential gene expression was determined using the 2^ΔΔCtmethod normalizing to the Ct-value of HPRT.

Samples were analysed using the eTAm-Seq technology in Laboratory 1 using an average of 12,450 AC per reaction. Digital PCR analysis was performed using a QX200 droplet dPCR system (Bio-Rad) with a C1000 Touch Thermal Cycler (Bio-Rad) at LGC. KRAS G12/WT and EGFR L858R/WT mutations were assessed using PrimePCR assays (Bio-Rad) and custom designed assays were used targeting NRAS A59T/WT and PI3KCA E545K/WT (S2A–S2C Table). Primers and BHQplus probes for custom assays were supplied by BioSearch and diluted in 1 x TE pH 8.0 (Sigma). Reactions (20 μL) were prepared (with 10% excess) and contained ddPCR Supermix for Probes with no dUTP (Bio-Rad), 20x primer/probe mix, 4 μL cfDNA extract (n = 1 per target mutation) with the remaining volume nuclease-free water (Ambion). Non-spiked Multiplex I cfDNA Reference Standards (32 ng/reaction) were analysed alongside the spiked extracts as controls (n = 3). Data was analysed using QuantaLife (Bio-Rad, version 1.6.6.0320) with classification of single positive, double positive and negative droplets as shown in S1A–S1E Fig. Copy number concentration was calculated based on a partition volume of 0.85 nL.

RT-qPCR data for 18080 protein-coding genes were generated in the context of the Sequencing Quality Control study (SEQC) (17) using PrimePCR assays (BioRad) (Supplemental Table 1). In order to define the ensemble of transcripts amplified by every individual qPCR assay, assays were re-mapped on the reference transcriptome (ensembl v75). Genes with a Cq-value between 11 and 32 were considered for further analysis. Cq-values were normalized using the global mean normalization strategy^{19 (link)}.

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)].