Peqgold trifast

Total RNA from the liver was isolated using the miRNA NucleoSpin® Kit (Macherey & Nagel, Düren, Germany) and quantified as described previously [32 (link), 55 (link)]. Total RNA from epididymal white adipose tissue (VAT) was isolated by optimized guanidine isothiocyanate/phenol-chloroform extraction using peqGOLD TriFast^TM (VWR International, Leuven, Belgium). The primers for the qRT-PCR were designed using Primer3 Input software version 4.1.0 (https://primer3.ut.ee/) and purchased from Eurofins MWG (Ebersberg, Gemany) (Table 1). The qRT-PCR was performed using the SensiMix™ SYBR No-ROX one step kit (Bioline, Luckenwalde, Germany) on a Rotorgene 6000 cycler (Corbett Life Science, Sydney, Australia). Target gene mRNA expression was calculated using an external standard curve and related to the housekeeper Rn18S (for liver) or the mean of Rn18S and Gt2b (for VAT).

For gene expression studies, the analysis was performed with the tissue of the peri-infarct region or cell lysates. Tissue samples were dissected using a stereomicroscopic approach, pooled, dissolved in peqGOLD Trifast^TM (VWR, Darmstadt, Germany), and homogenized. For cell culture samples, the supernatant was discarded, the cell layer was washed once with phosphate-buffered saline (PBS), and then peqGOLD Trifast^TM was placed on the cells. The extraction of total RNA was done following the manufacturers’ protocol, as previously described [47 (link)]. The RNA concentration and purity were measured with a NanoDrop 1000 device (VWR, Darmstadt, Germany).

We isolated RNA using peqGOLD TriFast^TM (30-2020, Peqlab, VWR, Radnor, PA, USA). Briefly, 500 µl peqGOLD TriFast^TM was added per sample and vortexed after addition of 100 µl chloroform (102445, Merck, Darmstadt, Germany) for at least 20 s. Approximately 200 µl colourless supernatant was transferred to a tube with 500 µl precooled isopropanol (20.842.330, VWR, Radnor, PA, USA) and incubated overnight at −80 °C. Samples were centrifuged for 30 min at 4 °C 13,000 rpm and the obtained pellet was washed twice with 80% ethanol (32205-1L‑M, Sigma-Aldrich, St. Louis, MO, USA). After the last washing step, the pellet was dried for at least 30 min and RNA concentration was determined using a NanoPhotometer (N60, Implen, Munich, Germany) after reconstitution in 20 µl RNase-free water (T143, Carl Roth, Karlsruhe, Germany).

Total RNA was obtained from cell lysates using EXTRACTME^® TOTAL RNA kit (blirt, Gdansk, Poland) and from tissue samples using peqGold TrifastTM (VWR International GmbH, Erlangen, Germany). Real-time RT-PCR reactions were performed in optical tubes containing cDNA template, each sense and anti-sense primer, and SsoFastTM EVAGreen Supermix (Bio-Rad Laboratories, Feldkirchen, Germany). Reaction mixture without cDNA was used as negative control. Relative quantification (2^−ΔΔCT) was performed using glyceraldehyde 3-phosphate dehydrogenase (Gapdh) housekeeping gene as reference. Mouse sense and antisense primer sequences were as follows: Gapdh: 5′-TGT TCC TAC CCC CAA TGT GT-3′, 5′-AGA GTG GGA GTT GCT GTT GA-3′, product size: 175 bp; Ccl2: 5′-ACT CAC CTG CTG CTA CTC AT-3′, 5′-TCA GCA CAG ACC TCT CTC TT-3′, product size: 138 bp; Il-3rα: 5′-TGG AGG AAG TCG CTG CTC TA-3′, 5′-CGT CAC CTC GCA GTC TTC AA-3′, product size: 111 bp; Tnf-α: 5′-ACC ACC ATC AAG GAC TCA-3′, 5′-AGG TCT GAA GGT AGG AAG G-3′, product size: 127 bp; Mc-cpa: 5′-CAT GGA CAC AGG ATC GAA TG-3′, 5′-TGC AGG TCC CCT GTA GAC AT-3′, product size: 152 bp; Mcpt4: 5′-ATC TTA TGG ACG CGG AGA TG-3′, 5′-GTG ACA GGA TGG ACA CAT GC-3′, product size: 185 bp; (all Eurofins Genomics, Ebersberg, Germany). The CFX 2.1 software and CFX Connect Real-Time PCR System of Bio-Rad Laboratories were used.

Gene expression study was performed using rtPCR technology (StepOnePlus Real-Time PCR System, Thermo Fisher, Waltham, MA, USA), KAPA SYBR^® FAST qPCR Master Mix (2X) Kit (Kapa Biosystems, Cape Town, South Africa). Briefly, tissue was rapidly homogenized using the Precellys Evolution tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), and extraction of total RNA was performed with peqGOLD TriFast^TM (VWR Life Science; Darmstadt, Germany). RNA samples (1 µg) were treated with DNase1 (Roche, Mannheim, Germany) to eliminate genomic DNA. Reverse transcription was conducted using the M-MLV RT-kit (Thermo Fisher Scientific, Waltham, MA, USA) and hexanucleotide primers. The qBase + software package Version 3.4 was applied to assign suitable reference genes and to estimate relative gene expression. GAPDH and HSP90 served as internal controls. Primer sequences (Table 1) were designed using Primer3web version 4.1.0 (https://primer3.ut.ee/; accessed on 1 July 2022) [50 (link)] or obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/; accessed on 1 July 2022) [51 (link)]. Finally, data of the experimental group (LL) were expressed as a fold of the control group (LD). Melting curves were analyzed to control for primer specificity.

RNA from organ tissue (stored in RNAlater® stabilizing reagent until further processing) was isolated using peqGold TriFastTM (Peqlab) and cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Primers and probes were chosen from the Universal ProbeLibrary (Roche). The fold change in gene expression was determined by the ΔΔCT method, using HPRT as housekeeping gene for calculation of ΔCT values and samples of naïve Myd88^+/− mice as calibrators.

RNA was extracted from hippocampus, cortex and brainstem using peqGOLDTriFastTM (PeqLab, 30-2040, Erlangen, Germany) reagent according to the manufacturer’s instructions. RNA was treated with DNaseI (Life Technologies, EN0521) to remove genomic DNA contamination. This procedure was followed by cDNA synthesis using Superscript III-reverse transcriptase (Life Technologies, 18,080,085) and Oligo (dT)₁₈-Primers (Thermo Scientific, SO132) at 50 °C for 1 h. cDNA was used as PCR template in an 1:10 dilution and each sample run in triplicate. Quantitative PCR was performed on StepOnePlus™ Real-Time PCR System (Applied Biosystems) using Express SYBR GreenER qPCR Supermix Universal (Life technologies, 1,178,401 K), 0.2 μM primer each on the DNA (primers as published by us before) with the following cycle conditions: primary denaturation at 95 °C for 3 min at 95 °C, 35 cycles with 30s at 95 °C, 30s at 60 °C and 30s at 72 °C followed by fluorescence measurement. Absolute quantification was performed for every single gene with three technical repeats per sample. Serial dilutions of plasmid controls with known molecule concentrations were used as a positive control and to generate standard curves. Expression of target genes were normalized using 36B4 (large ribosomal protein P0, RPLP0) as reference gene.