Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies
Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies
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DOI：
10.1017/s0031182016001025
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发表时间：
2016-10-01
期刊：
影响因子：
2.4
通讯作者：
Bowman, Alan S.
中科院分区：
文献类型：
--
作者：
Mcintosh, Catriona H.;Baird, John;Bowman, Alan S.
猫蚤是伴生动物的主要害虫，对动物保健产业具有重要意义。本研究的目的是开发采样和存储方案，并为基因表达研究寻找稳定的参考基因，以充分利用日益增长的猫眼蜂的分子知识体系。对成虫和幼虫样本的RNA完整性进行了评估，这些样本被穿孔或未被穿孔，并在常温下储存在RNAlater中。穿孔样品的RNA质量保持得最好，10天后几乎没有明显的降解。3天内未穿孔样品的RNA质量较差。通过四组比较(发育阶段、性别、取食状态和杀虫剂处理状态)，对10个候选参考基因的稳定性进行了评估。3-磷酸甘油醛脱氢酶(GAPDH)、60S核糖体蛋白L19(RPL19)和延伸因子-1(EF)在所有稳定性比较中排名较高，建议在类似条件下作为参考基因。仅使用这三个稳定的参考基因中的两个就足以进行准确的标准化。我们的结果对猫眼蜂基因表达研究的未来做出了重大贡献，描述了用于这种常见害虫的有效样本制备程序和参考基因。
The cat flea, Ctenocephalides felis, is a major pest species on companion animals thus of significant importance to the animal health industry. The aim of this study was to develop sampling and storage protocols and identify stable reference genes for gene expression studies to fully utilize the growing body of molecular knowledge of C. felis. RNA integrity was assessed in adult and larvae samples, which were either pierced or not pierced and stored in RNAlater at ambient temperature. RNA quality was maintained best in pierced samples, with negligible degradation evident after 10 days. RNA quality from non-pierced samples was poor within 3 days. Ten candidate reference genes were evaluated for their stability across four group comparisons (developmental stages, genders, feeding statuses and insecticide-treatment statuses). Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), 60S ribosomal protein L19 (RPL19) and elongation factor-1 (Ef) were ranked highly in all stability comparisons, thus are recommended as reference genes under similar conditions. Employing just two of these three stable reference genes was sufficient for accurate normalization. Our results make a significant contribution to the future of gene expression studies in C. felis, describing validated sample preparation procedures and reference genes for use in this common pest.