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Reference gene selection and RNA preservation protocol in the cat flea, Ctenocephalides felis, for gene expression studies

机译：猫跳蚤Ctenocephalides felis中的参考基因选择和RNA保留方案，用于基因表达研究

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摘要

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.
机译：猫蚤Ctenocephalides felis是陪伴动物上的主要害虫，因此对动物保健业具有重要意义。这项研究的目的是开发采样和存储协议，并为基因表达研究确定稳定的参考基因，以充分利用不断增长的猫科动物分子知识的发展。评估成年和幼虫样品的RNA完整性，这些样品被穿孔或未穿孔，并在室温下储存在RNAlater中。在穿孔的样品中，RNA质量保持最佳，10天后降解几乎可以忽略不计。未穿孔样品的RNA质量在3天内很差。在四个小组比较（发育阶段，性别，摄食状况和杀虫剂处理状况）中评估了十个候选参考基因的稳定性。甘油醛3磷酸脱氢酶（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均排名很高，因此推荐作为相似条件下的参考基因。仅使用这三个稳定参考基因中的两个就足以进行准确的标准化。我们的研究结果对猫屎肠球菌基因表达研究的未来做出了重大贡献，描述了经过验证的样品制备程序和用于该常见害虫的参考基因。

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