Computational protocol: A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos)

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Protocol publication

[…] At specific time points (t = 0.5, 1, 2, 4 and 7 days after introduction of the inoculated ducks, hereafter days post infection, dpi), five of the contact-infected ducks were randomly selected and sequentially euthanized humanely by mechanical disruption of the brainstem via the use of a CASH Poultry Killer (Accles & Shelvoke, Sutton Coldfield, UK). The five control ducks and three experimentally inoculated ducks were sequentially euthanized on day three, equivalent to 6 dpi for the inoculated ducks. Necropsies were performed immediately after death and the following tissues collected: blood; lung; spleen; two sections of the gastrointestinal tract (GI) corresponding to the distal jejunum, anterior to Meckel’s diverticulum (hereafter GI2), and the distal ileum, anterior to the joining of the caecum (hereafter GI4); and colon. Approximately 150 mg of each tissue was sliced into ~2 mm3 pieces, immediately snap frozen in liquid nitrogen and thereafter stored at -80°C until required for analysis.The sampling strategy outlined above and in was part of a larger experiment analysing the effects of LPAI infection on gene expression in mallards (unpublished data). As a first step, it was important to identify stable RGs. Thus, a subset of representative samples across treatment groups were chosen, as recommended in the QBase+ manual (https://www.biogazelle.com/sites/default/files/public_file/qbaseplus_manual.pdf). We selected 13–14 individuals comprising all uninfected ducks for which a sample was available (n = 4–5) and one third of the infected ducks (n = 9) spread across the time points (see for samples used). Only four uninfected samples were available for GI2 and GI4 due to labels peeling off tubes during storage in liquid nitrogen. RNA was extracted from the selected samples per tissue, using the RiboPure Kit (Ambion) following the manufacturers’ protocol, followed by DNase treatment. To confirm that DNase treatment successfully removed contaminating genomic DNA from RNA extracts, intronic primers were used to confirm absence of PCR amplification. Additionally, treated extracts were run on an agarose gel containing 2% bleach to visualise quality and integrity of RNA and confirm absence of DNA. Further details are provided as supporting information (). [...] cDNA was synthesised and amplified in a two-step process. Synthesis (step one) was performed via reverse transcription (RT), at a standard tissue-specific concentration, using Superscript III (Invitrogen, Carlsbad, CA) and random hexamers (Invitrogen). Further details are provided as supporting information (). cDNA was then amplified (step two) via qPCR to determine the transcription level of candidate RGs. Each reaction consisted of 5 μl 1:10 diluted cDNA, 10 μl iQ™ SYBR Green Supermix (BioRad, Carlsbad, CA), 150 mM each of the forward and reverse primers and DNase free H2O to a final volume of 20 μl. Negative controls, comprising cDNA-free reactions and RT-minus reactions, were included for every gene and tissue combination (see also ). qPCR reactions were run on a LightCycler480 (Applied Biosytems, Foster City, CA) with the following cycle for all genes: an initial denaturation of 95°C for three minutes, followed by 45 cycles of 95°C for 10 seconds (s), and 58°C for 30 s, with data acquisition at the end of each elongation step. Immediately following qPCR, a melt analysis was performed (95°C for 10 s with a ramp rate of 4.4°C/s, 58°C for one min with a ramp rate of 2.2°C/s, followed by a slow incremental increase in temperature to 97°C with a ramp rate of 0.11°C/s and continual acquisition), finally samples were cooled to 40°C for 30 s. All qPCR reactions were run in duplicate.Primers () were designed, with reference to the duck genome [], using the primer design tool in NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). To confirm amplification of the desired products, 2–5 PCR products per gene were sequenced (Eurofins MWG Operon, Elmsburg, Germany) and blasted (Nucleotide BLAST, NCBI) to confirm homology with the expected genes. Our choice of candidate RGs to test was informed in part by those used previously in chicken [, ], as well as discussions with colleagues with experience in gene expression analysis. [...] The algorithms GeNorm [] and NormFinder [] were used to quantify stability of the eleven putative RGs. GeNorm calculates an internal control gene stability measure M for each gene (M value), by computing the average pairwise variation of a particular gene with all others under consideration []. Genes with the lowest M values have the most stable expression, with values under 0.5 indicative of acceptably stable expression. GeNorm ranks genes from least to most stable by stepwise exclusion of the least stable gene and repetition of the analysis until only two most stable genes remain. Furthermore, GeNorm calculates the number of genes required for accurate normalisation (V value) by comparing the coefficient of variation of the n most stable gene(s) with the n+1 most stable genes (Vn/n+1) whereby a V value below 0.15 indicates that n genes are sufficient for stabilisation []. We implemented GeNorm analyses in QBase+ []. NormFinder calculates an internal stability value for each gene based on expression variance, and also provides the option to consider variance between experimental subgroups [], which we set as the variance in expression between infected and uninfected individuals. NormFinder requires input data to be linearized, which we did by transforming raw Ct values using the formula RQ = 1 / (2(Ct,sample−Ct,min)) where RQ is the relative quantity, Ct,min is the lowest Ct value obtained for any sample for that gene and tissue combination, and Ct,sample is the Ct value of the sample being standardised []. We implemented NormFinder analyses in Excel using a freely available macro (http://moma.dk/normfinder-software). All data was plotted in GraphPad Prism v.6.03 (GraphPad Software Inc, San Diego, CA). […]

Pipeline specifications

Software tools qbase+, Primer-BLAST, NormFinder
Application qPCR
Organisms Anas platyrhynchos, Homo sapiens
Diseases HIV Infections