Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae)
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Abstract
Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors in question by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. These reference genes can be used to study the hormonal regulation of reproduction and parental care in Lethrus apterus in the future.
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2017. Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae) PeerJ Preprints 5:e3104v1 https://doi.org/10.7287/peerj.preprints.3104v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Nikoletta A Nagy conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Zoltán Németh conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper.
Edit Juhász conceived and designed the experiments, performed the experiments, wrote the paper, reviewed drafts of the paper.
Szilárd Póliska conceived and designed the experiments, performed the experiments, wrote the paper, reviewed drafts of the paper.
Rita Rácz conceived and designed the experiments, performed the experiments, wrote the paper, reviewed drafts of the paper.
András Kosztolányi conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper.
Zoltán Barta conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers):
Sample collection was approved by the National Inspectorate for Environment Protection and Nature Conservation.
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The housekeeping gene sequences described here will be available via GenBank accession numbers KY786273 to KY786283 as soon as the accession numbers appear in print or on 1st December 2017. For reviewers, this information is attainable in Supplemental_file_1.
Data Deposition
The following information was supplied regarding data availability:
Sequences were accepted by GenBank and will be released as soon as the accession numbers appear in print or 1st December 2017.
Funding
The study was financed by NKFIH Grant (No. K112670). Zoltán Barta was supported by NKFIH Grant (No. K112527). András Kosztolányi was supported by János Bolyai Research Scholarship of the Hungarian Academy of Sciences. During the preparation of the manuscript, Zoltán Németh was supported by a fellowship from the National Research, Development and Innovation Office of Hungary (No. NKFIH PD 121013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
