A novel universal probe library quantitative reverse transcription polymerase chain reaction method to profile immunological gene expression in blood of captive beluga whales (Delphinapterusleucas)
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Abstract
Cytokines are fundamental for a functioning immune system, and thus, potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a novel probe-based quantitative gene expression assay for immunological assessment of captive beluga whales ( Delphinapterusleucas ) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.
Cite this as
2017. A novel universal probe library quantitative reverse transcription polymerase chain reaction method to profile immunological gene expression in blood of captive beluga whales (Delphinapterusleucas) PeerJ Preprints 5:e2735v1 https://doi.org/10.7287/peerj.preprints.2735v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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This is a submission to PeerJ for review.
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Supplemental Information
Cq values of the tested samples
Cq values of 6 target genes and 2 reference genes of the 12 healthy samples and 4 non-healthy samples.
Additional Information
Competing Interests
Hsiao-Kuan Ho is an employee of Hi-Scene World Enterprise Co Ltd., 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
Author Contributions
Ming-An Tsai conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper.
I-Hua Chen conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Jiann-Hsiung Wang conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Shih-Jen Chou conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Tsung-Hsien Li performed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Ming-Yih Leu performed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Hsiao-Kuan Ho performed the experiments, contributed reagents/materials/analysis tools, reviewed drafts of the paper.
Wei Cheng Yang conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
Animal protocol was reviewed and approved by the Council of Agriculture of Taiwan (Approval number 1020727724).
Data Deposition
The following information was supplied regarding data availability:
The raw data has been supplied as a supplementary file.
Funding
This work was supported by Ministry of Science and Technology, Taiwan [grant number MOST 104-3113-E-002-012]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.