Itaq universal syber green supermix

Colon tissue was transferred to a 2 mL reinforced tube (Precellys Lysing Kit) containing 1.4 mm ceramic beads (zirconium oxide, Precellys Lysing Kit) and 1 ml TRIzol Reagent (Invitrogen). Tissue was disrupted in a Precellys homogenizer (Bertin instruments) for 2 × 30 s at 6500 rpm. Nucleic acids were extracted in 0.2 volume of chloroform (Sigma) and purified by precipitation in 0.5 volume of isopropanol (Sigma). RNA pellets were dried and resuspended in 40 µL of RNAse-free water. Nucleic acid concentrations were determined by measuring absorbance at 260 nm using a NanoDrop spectrophotometer (ThermoScientific). One microgram of total RNA was treated for reverse transcription with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions. Quantitative PCR was performed with iTaq Universal SYBER Green Supermix (Biorad) and the primer sets listed in Supplementary Table 3. The thermal cycling conditions comprised 30 s at 95 °C and then 40 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by a standard melting curve analysis. Gene expression values were calculated based on the ΔΔCt method, ΔCt values were calculated using the Ct values from the amplification of a reference gene (GAPDH, housekeeping gene). Quantitative real-time PCR was performed on the CFX96 PCR system (Biorad).

Total RNA was extracted with TRI Reagent (Sigma-Aldrich) from 15 to 25 adult of each genotype per biological replicate. The reverse transcription was performed on 1 μg of RNA by using the RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). cDNA was used as template in PCR reaction with three 10 μl technical replicates of each biological sample. qPCR was performed on an iQ5 Real Time PCR detection system (Biorad) using iTaq Universal Syber Green supermix (Biorad). The amount of RNA detected was normalized to that of the house keeping gene rp49. Primers for Diptericin and rp49 genes are listed in the Supplementary Table 1. Relative gene expression levels between control and experimental samples was determined using the ΔΔCT method. Each experimental sample was compared to each wt sample.

RNA from 5x10^6 splenocytes was extracted with the RNeasy Mini Kit (BioRad) and converted to cDNA using the Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific). Quantitative reverse transcriptase polymerase chain reation (RT-qPCR) using iTaq Universal Syber Green Supermix (BioRad) and respective primers (Supplementary Table 1) was performed on cDNA to quantify the expression of Ifih1, Mx1, Ifit1, Hprt, and Oas1 genes. The plates were run on a Roche LightCycler 480 II. For ΔC_t values, gene expression was normalized to Hprt. Fold differences in mRNA expression (relative expression) were determined using the equation [2^(-ΔΔCt)].

GPR21 gene expression was quantified by a real-time qPCR. Total RNA was extracted by using TRI Reagent^® (Sigma, Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s instructions. Complementary DNA synthesis and quantitative real-time PCR (q-PCR) reactions were performed on cell samples as previously described [57 (link)]. mRNA levels were measured by a q-PCR, using the SYBR^® green method as described [58 (link)] The amplification mix was prepared using iTaq Universal Syber Green SuperMix (Biorad Laboratories, Berkeley, CA, USA) following the manufacturer’s instructions and the real-time PCR was performed using Miniopticon ThermoCycler Instrument (Biorad Laboratories, Berkeley, CA, USA). A real-time amplification of human GPR21 was carried out using the following set of primers:
Human GPR21 FW 5′-TTTTCCACTGGGGCAAACCT-3′;
Human GPR21 RV 5′-TTGGCAGATGCGGAAGATGT-3′.
The housekeeping human gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified in parallel, in all amplification sets, using the following set of primers:
Human GAPDH FW 5′-TGGTATCGTGGAAGGACTCATGAC-3′;
Human GAPDH RV 5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′.