Rnase free water

IL gene expression (IL-6, IL-8, and IL-10) in ileal tissue was quantified using RNA extraction used an RNeasy Mini Kit (Catalogue no.74104, Qiagen). A 30 mg of tissue sample was homogenized and processed as described by the manufacturer. IL-6, IL-8, and IL-10 mRNA were amplified and quantified using a real-time PCR machine (Stratagene MX3005P, Agilent Technologies Company, USA). Primer sequences are listed in Table-1 [23 (link),24 ]. The housekeeping gene 28S rRNA was used as a constitutive control for normalization. The reaction mixture volume was 25 μL made up with 12.5 μL 2x QuantiTect Probe RT-PCR Master Mix (catalog No.204443, Qiagen), 0.5 μL of 20 pmol solution of each primer, 0.125 μL probe (30 pmol), 0.25 μL QuantiTect RT Mix (RevertAid Reverse Transcriptase) (Qiagen), 8.125 μL RNase-free water (Sedico, Egypt), and 3 μL template RNA. The real-time PCR cycling conditions were reverse transcription (50°C, 30 min), primary denaturation (94°C, 10 min), and then amplification (40 cycles) with secondary denaturation (94°C, 15 s), annealing, and extension (60°C, 1 min). Amplification curves and Ct values were determined using Stratagene MX3005P software, Agilent Technologies Company). The Ct value of each sample was compared with the positive control as described for the “DDCT” method [25 ].

Total RNA was extracted from the liver, distal intestine and pyloric caeca from 5 fish from each tank (n = 15) using RNAzol RT reagent (Merck KGaA), eluted in 20 µL of RNase-free water (Qiagen), and then stored at −80 °C until use [83 (link)]. A NanoPhotometer P-Class (Implen, München, Germany) was used to determine final the RNA concentration of each sample. RNA integrity was verified by GelRed^TM staining of 28S and 18S ribosomal RNA bands on 1% agarose gel. A cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Bio-Rad, Milano, Italy) from 1 μg of RNA.

We isolated total RNA from individual wasp brains. Wasp heads were first freeze-dried for 60 min at 0.315 Torr on a 2.5 L Benchtop Labconco Freeze Dryer. Thereafter, heads were placed on a dissection plate on dry ice and we removed the outside cuticle with a fine scalpel. Exposed brains were separated from the head capsule, maintained in dry ice and immediately frozen at − 80 °C. To isolate total RNA we used the RNeasy Mini kit (Qiagen) and followed standard manufacturer instructions. We added a step of DNase treatment (QIAGEN) to remove possible contamination with genomic DNA. We checked quality and quantity of RNA samples with a spectrophotometer (NanoDrop 2000 and Qubit instruments). Only samples that passed quality check (260/280 ratio > 2) were selected for follow-up analyses. These samples were dried in a Labconco FreeZone 2.5L Lyophilizer, and resuspended in RNase-Free Water (QIAGEN) to a concentration of 40 ng/µl.

RNA was extracted using the RNeasy kit (Qiagen, Crawley, UK), according to the manufacturer’s instructions. For cDNA synthesis, 300–500 ng RNA was used in combination with 0.5 μg random primers (Roche, West Sussex, UK), 40 U RNaseOUT 0.5 μM dNTPs (Invitrogen, Paisley, UK) 1× RT Buffer (Fermentas, Cambridge, UK) and RevertAid reverse transcriptase (Fermentas, Cambridge, UK) made up to a final volume of 20 μl using RNase-free water (Qiagen, Crawley, UK). Reactions were carried out in a thermocycler with conditions—25 °C for 10 min, 42 °C for 60 min and 70 °C for 10 min. One microlitre cDNA per well on a 96-well plate (Roche) was used for RT-qPCR with SYBR Green reagent and remaining cDNA stored at −80 °C. RT-qPCRs were performed using a LightCycler 480 Instrument II (Roche, West Sussex, UK). Gene expression was normalised to Hprt and relative expression calculated by the ΔΔC_T method [40 (link)]. Each RT-qPCR contained 1× buffer, 0.4 mM dNTPs, 50 μM primers (Additional file 1: Table S1), 0.01 U Taq DNA polymerase (Invitrogen, Paisley, UK) and nuclease-free water (Qiagen, Crawley, UK). Four primer sets for Dnmt1, Dnmt3a, Dnmt3b [47 (link)] and Hprt [13 (link)] were used. The general thermocycler conditions are as follows—94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 63 °C for 1 min, 72 °C for 1 min with a final elongation step of 72 °C for 4 min.

Cell pellets obtained after centrifugation of exponentially growing cultures were immediately re-suspended in 1 ml of 60°C hot TriReagent (Sigma-Aldrich, Steinheim, Germany) and transferred into a 2 ml cryovials containing acid washed glass beads (Sigma-Aldrich, Steinheim, Germany). As for DNA extraction, cell lysis was achieved with a Bio101 FastPrep instrument (Thermo Savant Illkirch, France) run at maximum speed (6.5 ms⁻¹) for 45 s. RNA isolation was then performed as described in [64 (link)]. In brief, after thawing the cell lysate on ice, 200 μl of pure chloroform was added to each sample. The sample was vortexed thoroughly and incubated for 10 min at room temperature. The aqueous phase was separated by centrifugation and transferred into a new vial together with 100% isopropanol and incubated at -20°C to precipitate the RNA. The pellet was collected by centrifugation at 4°C, washed with 70% ethanol, air-dried and re-suspended in RNase-free water (Qiagen, Hilden, Germany).
Only RNA samples with high quality (OD 260/280 > 2 and OD260/230 > 1.8), determined with a NanoDrop ND-100 spectrometer (PeqLab, Erlangen, Germany), and high RNA integrity, checked with the Agilent RNA Nano Chip Assay (Agilent, Santa Clara, USA), were used for transcriptome sequencing.

Total RNA of Huh7 cells was isolated using TRIZOL (Fischer Scientific, Reinach, Switzerland) according to the manufacturer’s instructions. The dried RNA pellet was resuspended in 20 µL of RNAse free water (Qiagen, Basel, Switzerland). Total RNA from zebrafish tissue (liver or zebrafish eleuthero-embryos) was extracted using RNeasy Mini Kit (Qiagen, Basel, Switzerland) according to the manufacturer’s instructions. RNA concentrations and purity were measured spectrophotometrically (Nanodrop ND-1000, Witec AG, Lucerne, Switzerland using ND1000 software, version 3.8.1), and single strand cDNA synthesis and RT-qPCR were performed as previously described [20 (link)].