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

Abstract

Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard—a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used β-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. β-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.

A divergent hepatitis D-like agent in birds

“…Avian viral transcripts were highly abundant in the library, largely representing influenza A virus. RNA from an avian HDV-like agent was ten-fold less abundant than that of influenza A virus, but was more abundant than RNA from the host reference gene RPS13 that is stably expressed in ducks (10) (Fig 1B). Exogenous duck hepatitis B virus (DHBV) was not identified in this meta-transcriptomic library.…”

“…To compare viral abundance to that of the host, a blast database was created containing Ribosomal Protein S13 (RPS13) from both Mallard (taxid: 8839) and Chicken ( Gallus gallus ) (taxid: 9031), which has found to be stably expressed in the Mallard ( Anas playrhynchos ) lower gastrointestinal tract (10).…”

Sustained RNA virome diversity in Antarctic penguins and their ticks

“…The abundance of RSP13, a stably expressed host reference gene in the avian colon [47], was similar across all penguin libraries, yet with lower abundance in the Adelie penguins (Fig. 2c).…”

The Intestinal Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks

“…Mixes were prepared according to the manufacturer instructions (QuantiFast SYBR Green PCR, Qiagen, Toronto, Canada) with 1µl of 1:20 diluted cDNA and a final concentration of 1µM of each primer (Table.1). Relative quantification was carried out using the 2 −ΔΔCT method (53) and the geometric means of two couples of housekeeping genes validated in specific duck tissues: RPL4/RPL30 and RPL4/SDHA for ileum and lung samples, respectively (54). Quantification of influenza virus nucleic acid load in tissues was performed in 96-well plates with 20µL final volume according to the manufacturer instructions (QuantiFast SYBR Green PCR, Qiagen, Toronto, Canada), 2µl of cDNA and a final concentration of 1µM of each primer.…”