Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes
- Published
- Accepted
- Subject Areas
- Ecology, Microbiology
- Keywords
- qRT-PCR normalization, gene expression, Suillus, heavy metal tolerance, intraspecific variation
- Copyright
- © 2016 Ruytinx et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2016. Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes. PeerJ Preprints 4:e2125v1 https://doi.org/10.7287/peerj.preprints.2125v1
Abstract
Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.
Author Comment
This is a preprint submission to PeerJ Preprints.
Supplemental Information
Primer efficiency calculation
Raw Cq's of cDNA (mix sample) dilution series. Linear regression statistics and efficiency calculation.