Ssoadvanced universal sybr supermix

Tumors from PyMT/p11-WT and PyMT/p11-KO mice at 20 weeks of age were excised, snap frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using Trizol (Invitrogen, Thermofisher Scientific, Waltham, MA, USA) and the PureLink RNA kit (Invitrogen, Thermofisher Scientific, Waltham, MA, USA ) with DNase treatment. Equal amounts of RNA (0.5 µg) were reverse transcribed using iScript (BioRad, Mississauga, ON, Canada); qPCR reactions were performed with SsoAdvanced Universal SYBR Supermix (BioRad, Mississauga, ON, Canada) and gene-specific mouse primers (Table S2) on a CFX96 or CFX384 Touch Real-Time PCR Detection system (BioRad, Mississauga, ON, Canada). Standard curves for each primer set were generated, and primer efficiencies were incorporated into the CFX Manager software (BioRad, Mississauga, ON, Canada). mRNA expression of all samples was calculated using the ΔΔct method, with gene of interest made relative to two reference genes (rlp10 and PyMT) and an indicated control sample. Relative mRNA expression was log−2 transformed prior to plotting and statistical analysis.

The expression level of genes for MUC2 and antioxidant enzymes (i.e., SOD, CAT, GPX) in the ileal tissue was determined using the isolated RNA samples obtained from the section.
First, cDNA was synthesized from the RNA samples using iScript Reverse Transcription Supermix (BioRad, USA). A total of 4 µL of 5 × iScript reaction mix (RT/No-RT) was added to each calculated sample. Primers used in the qRT-PCR analysis were designed in our laboratory and are presented in Table 3. The qRT-PCR was performed using SYBR green supermix. Bio-Rad CFX connects with a working sample of the 3 µL of each cDNA sample, 10 µL of SSO Advanced Universal SYBR Supermix (Bio-Rad), 1 µL (0.5 µL forward and reverse) of each primer, 3 µL of each cDNA template, 6 µL of free DNAse/RNAse water, totaling 20 µL which was distributed in triplicate to a 96-well plate and sealed using BioRad Microseal ‘B’ seal (an optically clear heat seal), centrifuged and placed inside a CFX connect machine running at a temperature of 60 °C. ΔΔCT was used in calculating the fold changes for the upregulation or downregulation of genes.

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)].

Isolation of total RNA from rabbit tissues was performed using TRIzol reagent (Life Technologies, Paisley, UK) and a RNeasy Mini Kit (Qiagen, Hilden, Germany), both according to the manufacturers’ instructions. Complementary DNA (cDNA) synthesis was carried out using the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) and quantitative real-time RT-PCR (qRT-PCR) amplification and detection were carried out using SsoAdvanced Universal SYBR^®® Supermix and the CFX96 Two-Color Real-Time PCR Detection System (both Bio-Rad Laboratories). Specific PCR primers for rabbit target genes were designed on sequences available at National Center for Biotechnology Information (NCBI) GenBank (https://www.ncbi.nlm.nih.gov/) or Ensemble Genome (http://www.ensembl.org). The 18S ribosomal RNA subunit was quantified with a predeveloped assay (Hs99999901_s1, Life Technologies) and used as the housekeeping gene for the relative quantitation of the target genes according to the comparative threshold cycle (Ct) 2^−ΔΔCt method [64 (link)], with some modifications. In detail, we used the untreated group (RD) as the calibrator in each analysis, so that by definition the calculations would provide the fold-change of the treated group relative to RD. Data are reported graphically as a percentage over the RD group, whose mean was set at 100% for direct comparison of each measurement.

For quantitative real-time PCR (qRT-PCR) analysis, total RNA was extracted from the experimental samples using Total RNA Mini Plus Concentrator (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. One microgram of DNase-treated RNA was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Pittsburgh, PA, USA). Triplicate samples for quantitative PCR were run in a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). SsoAdvanced Universal SYBR Supermix (Bio-Rad, Hercules, CA, USA) was used for detection. mRNA expression in each sample was determined after normalization to Gapdh, 18S ribosomal RNA, or Hprt. Gene expression values are expressed as fold changes relative to the control using the ΔΔCt method. Sequences of primers that were used for qRT-PCR are listed in Table S1.

Cells were seeded in six‐well plates and exposed to a panel of SiO₂ NPs at 1 and 10 µg/mL for 24 h. After exposure, cells were collected and washed with PBS before processing for RNA isolation. RNA was isolated using the QIAGEN RNeasy Mini Kit by following the manufacturer’s protocol. The quality and yield of RNA was checked using NanoDrop (ThermoScientific). cDNA was synthesized using iScript^TM Reverse Transcriptase Kit (Bio‐Rad) using a thermal cycler (Bio‐Rad). RT‐PCR was performed using SYBR‐Green-based 96‐well primePCR custom plates (Bio‐Rad) for the following genes: APOE (qHsaCED0044297), SPNS2 (qHsaCID0008369), and GAPDH (qHsaCED0038674). Each RT‐PCR reaction contained 1 µL of cDNA, 1x SsoAdvanced universal SYBR supermix (Bio‐Rad), and 1x PrimePCR assay dried in a well. RT‐PCR was run using the AB7500-Standard RT‐PCR (Applied Biosystems) at the following conditions: activation at 95 °C for 2 min, 40 cycles of denaturation at 95 °C for 5 s, and annealing/elongation at 60 °C for 30 s. The fold change in the gene expression was obtained by calculating the ΔΔCt value with respect to GAPDH as reference.

For all transcript expression analyses by QPCR, cells were collected in TRIzol and total RNA was purified using a PureLink RNA kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Equal amounts of harvested RNA were reverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad, Saint Laurent, QC, Canada) as per the manufacturer’s instructions. QPCR was performed using SsoAdvanced Universal SYBR Supermix (Bio-Rad) and transcript-specific primers (primer sequences are listed in Table S1) as per the manufacturer’s recommended protocol using a CFX96 Touch RealTime PCR Detection System (Bio-Rad). Primer efficiencies, determined by standard curves of diluted cDNA samples, were incorporated into the CFX Manager software (Bio-Rad). Gene expression of all samples was calculated relative to two or three reference genes as indicated in the figure legend and relative to the negative control (Table S1).