Computational protocol: Early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles

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Protocol publication

[…] RNA extractions from the proximal third of tracheal and renal samples of experimentally infected chickens were performed using TRIzol Reagent (Invitrogen) followed by RNA purification using RNeasy Mini Kit (Qiagen). The extracted RNA purity and quantification were estimated by 260nm ultraviolet absorbance and readings at 260/280nm, respectively. RNA quality was verified with Agilent RNA 6000 Nano Kit (Agilent Technologies) in an Agilent 2100 Bioanalyzer instrument (Agilent Technologies) for determination of RIN (RNA Integrity Number) or by RNA analysis in 1% gel electrophoresis.All tracheal and renal samples were tested for IBV viral load by RT-qPCR (hydrolysis probe system) using AgPath-ID One step RT-PCR kit (Ambion), primers and LNA-probe (5´FAM-3´BHQ1, IDT) for amplification of the 3´UTR genome region of IBV, as described []. Cq (Cycle quantification) results were used to calculate the Log of RNA copies (Log10) using the linear equation from a standard curve. Samples presenting Cq ≤ 36 were classified as positive for IBV.Two-step RT-qPCR was used for the relative quantification of gene expression in the tracheal samples. cDNAs were synthesised according to instructions provided with High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and 1μL (500 ng/μL) of Oligo(dT) primers (IDT). The PCR reactions contained 20 ng of cDNA, 7.5 μL of 2X Quantifast SYBR Green Master Mix (Qiagen), and 3 μM of each primer () in a final volume of 15 μL. Amplification included a pre-incubation step at 95°C for 5 min, followed by 40 cycles of 95°C for 15 seconds and 60.0°C for 35 seconds. After amplification, a melting curve analysis was performed by raising the incubation temperature from 65°C to 95°C in 0.2°C increments with a hold step of 1 sec at each increment. All oligonucleotides used were designed using Primer3 [http://frodo.wi.mit.edu] software, spanning exons according to gene sequences from Ensembl [http://ensembl.org] and mRNA sequences deposited in GenBank. Except for IFNα and IFNβ which are intron-less mRNAs, then, residual genomic DNA was digested with 2 μL DNAse I (Promega) before cDNA synthesis. Efficiency of each specific Real-time PCR was calculated using two-fold serial dilutions of cDNA pooled from all tested animals, and Cq values plotted into the linear equation.The relative expression of all tested genes () in tracheal samples of IBV-infected chickens was quantified as the fold change relative to the non-infected group (negative control), and the gene expression from each sample were standardised using the Cq value of the TOP2B/HPRT1 constitutive reference genes for the same sample []. The stability of the reference genes was tested using four candidates (TOP2B, HPRT1, GAPDH and Histone H3) and the best genes were selected using Bestkeeper and Normfinder softwares. […]

Pipeline specifications

Software tools Primer3
Databases ag.db
Application qPCR
Organisms Gallus gallus, Infectious bronchitis virus