Hydroxymethylbilane Synthase

RNA isolation from the tibiae from experiment A was performed by pulverizing the diaphysis with a freezer mill in liquid nitrogen (Spex Certiprep 6750 FreezerMill, Spex Certiprep, Metuchen, NJ, USA). RNA was extracted from bone powder with Trizol for 1 hour at 37°C and re-extracted once with phenol and once with chloroform. Next, the samples were extracted again with Trizol in accordance with the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). RNA extracts were incubated with DNAse to eliminate DNA contamination and stored at –80°C. The absorption at 260 and 280 nm was measured to determine the amount of RNA. A total of 100 ng of total RNA was transcribed into cDNA in a 20 μl mixture containing M-MLV Reverse Transcriptase (Promega, Fitchburg, WI, USA). All RNA samples were assayed in triplicate. Quantitative PCR was performed in a BioRad iCycler Real-time PCR system with three μl of each cDNA sample, 300 nM of each primer and SYBR Green Supermix (BioRad, Hercules, CA, USA) in a total volume of 25 μl. Table 1 lists the primer details of the genes of interest, which were Fgf23, Mepe, Dmp1, Phex, Cyp27b1 and Vdr and the housekeeping genes, which were hypoxanthine guanine phosphoribosyl transferase (Hprt) and porphobilinogen deaminase (Pbgd). After the PCR run a melting curve was conducted from 50°C to 95°C to check the specificity of the reactions. Mean Ct-values of each RNA sample assayed in triplicate were used. Gene expression for the genes of interest was normalized for the housekeeping genes ΔCt = (Ctgene of interest−Ct_{housekeeping gene)} and expressed as fold difference from the average of the housekeeping genes (2^-ΔCt) [28 (link)].

Total RNA was extracted from the piglet jejunal mucosal samples using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. DNase treatment was performed to remove contaminating DNA using the TURBO DNA-free™ DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the recommended protocol. The quantity and quality of the RNA were evaluated using a Nanodrop ND 1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA) and agarose gel electrophoresis, respectively. A total of 1000 ng of RNA was then converted into complementary DNA using the High-Capacity RNA-to-cDNA™ Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Duplex Real Time PCR reactions contained 2 µL cDNA and 8 µL mix containing primers, probe (Additional file 1: Table S1) and 2X TaqMan Mastermix, and were run in triplicate on the Applied Biosystems QuantStudio™ 7 Flex Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with the following thermocycler settings: 50 °C for 2 min, 95 °C for 2 min and 40 cycles of 95 °C for 1 s and 60 °C for 20 s. Hydroxymethylbilane synthase (HMBS) was used as housekeeping gene. The following genes were selected and analysed: innate immune signal transduction adaptor (MyD88), nuclear factor kappa B subunit 2 (NFKB2), Occludin (OCLN), Tight junction protein 1 (ZO-1), Mucin 13 cell surface associated (MUC13), glutathione peroxidase 2 (GPX-2), Claudin-4 (CLAUD4), Claudin-3 (CLAUD3), polymeric immunoglobulin receptor (PIGR), branched chain amino acid transaminase 2 (BCAT2), ornithine decarboxylase 1 (ODC1), solute carrier family 6 (neutral amino acid transporter), member 19 (SLC6A19), solute carrier family 7 member 9 (SLC7A9), solute carrier family 1 member 5 (SLC1A5), solute carrier family 38 member 2 (SLC38A2).
QuantStudio Design and Analysis Software v2.5 (Thermo Fisher Scientific, Waltham, MA, USA) was used for determining the gene expression cycle threshold (Ct) values. For each sample the Ct value of the HMBS gene was subtracted from the Ct value of the target gene (ΔCt). The average ΔCt value of the reference animals was then subtracted from the ΔCt value of all the samples (ΔΔCt). The expression of the target gene was given as fold change calculated by 2^−ΔΔCt.

Cell culture or lung RNA samples were collected and incubated in RNAlater reagent (Ambion). To extract total RNA, lungs were homogenized in 2 mL of RLT lysis buffer (Qiagen) supplemented with 1% β-mercaptoethanol using the GentleMACS Dissociator system (Miltenyi Biotec) following the protocol proposed by the manufacturer. RNA was purified from supernatants using the rNeasy minikit reagent (Qiagen), as previously described. To generate cDNAs from the purified RNAs, a reverse transcription (RT) reaction was carried out using the High Capacity cDNA RT kit reagent (Applied Biosystems). To verify the sequence of the mutations introduced into the viral genome, 4 μL of the cDNA generated in the RT reaction was used as a template in a PCR using the enzyme Taq polymerase (Invitrogen). Cellular mRNA expression was analyzed by quantitative reverse transcription-quantitative PCR (RT-qPCR), using TaqMan technology with commercial probes (ThermoFisher Scientific) (Table 2). In all cases, the reaction was performed with the FastStart Universal Probe Master reagent (Rox) (Roche). qPCRs were performed in a 7500 Real Time PCR System (ThermoFisher Scientific). qPCR data were analyzed using the 7500 software v2.0.6 (ThermoFisher Scientific). All experiments met the recommendations for analysis of gene expression by RT-qPCR (MIQE) (79 (link)). Each result is the average of three independent experiments in which each sample was tested in triplicate. The relative quantification of gene expression was performed from the mean values of CT (cycle in which the amplification curve cuts at the threshold level using the 2^−ΔΔCT method) (80 (link)). The level of 18S rRNA was used as an endogenous control to normalize cellular mRNA levels in mouse lungs, while hydroxymethylbilane synthase (HMBS) was used to normalize cellular mRNA levels in cell cultures.