Tsai Ming-An, Chen I-Hua, Wang Jiann-Hsiung, Chou Shih-Jen, Li Tsung-Hsien, Leu Ming-Yih, Ho Hsiao-Kuan, Yang Wei Cheng
Department of Biology, National Museum of Marine Biology and Aquarium, Pingtung, Taiwan.
College of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan.
PeerJ. 2017 Sep 27;5:e3840. doi: 10.7717/peerj.3840. eCollection 2017.
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 probe-based quantitative gene expression assay for immunological assessment of captive beluga whales () 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 RNA™ 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.
细胞因子对于正常运作的免疫系统至关重要，因此有可能作为动物健康的重要指标。使用定量逆转录聚合酶链反应（qRT-PCR）对mRNA进行定量是一种成熟的免疫学技术。它特别适用于检测那些没有单克隆抗体的蛋白质的表达。在本研究中，我们开发了一种基于探针的定量基因表达检测方法，用于圈养白鲸（白鲸是全球水族馆中最常见的鲸类物种之一）的免疫学评估。选择了六个与免疫相关的基因（IL-2Rα、-4、-10、-12、TNFα和IFNγ）进行分析，并使用两个表达稳定的经过验证的管家基因（PGK1和RPL4）作为参考基因。从四只健康状况不同的动物身上采集了16份血样，并保存在RNA™溶液中。这些样本用于RNA提取，随后进行qRT-PCR分析。通过相对定量，使用比较Cq法并结合扩增效率和两个参考基因对基因转录本进行分析。临床健康动物样本中每个基因的表达水平呈正态分布。在从两只临床不健康动物采集的四个样本中，发现IL-2Rα、IL-4、IL-12、TNFα和IFNγ的转录异常值。该检测方法有潜力识别由健康问题导致的免疫系统偏离正常状态的情况。此外，了解圈养鲸类的免疫状态有助于训练员和兽医在疾病发作前实施预防措施。
