Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA; Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; Laboratório de Bioquímica de Artrópodes Hematófagos, Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, RJ, Brazil.
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.
Ticks Tick Borne Dis. 2023 Nov;14(6):102251. doi: 10.1016/j.ttbdis.2023.102251. Epub 2023 Sep 12.
Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.
对基因表达的转录调控的研究对于理解生物体生理或细胞状态的变化至关重要。为了获得有关 mRNA 量和基因表达水平估计的可靠数据，必须使用一个或多个内参基因对靶基因进行标准化。然而，将组成型基因用作内参基因是有争议的，因为它们的表达模式有时比以前认为的更为复杂。在包括蜱在内的各种节肢动物载体中，已经通过研究不同组织和生命阶段的基因表达来鉴定了几种组成型基因。牛蜱 Rhipicephalus microplus 是几种病原体的主要载体，广泛分布于全球热带和亚热带地区。蜱的发育生理学是研究的一个重要方面，特别是胚胎发生，其中发生了许多重要的发育事件，因此，鉴定稳定的内参基因对于解释可靠的基因表达数据至关重要。本研究旨在鉴定和选择 R. microplus 管家基因，并评估它们在胚胎发生过程中的稳定性。分子分析中用作内参的内参基因是基于先前的研究选择的。通过定量 PCR (qPCR) 筛选这些基因，并在胚胎发生过程中测试它们的基因表达稳定性。结果表明，在胚胎发生过程的不同时间点，参考基因的相对稳定性有所不同。GeNorm 工具显示，伸长因子 1α (Elf1a) 和核糖体蛋白 L4 (Rpl4) 是最稳定的基因，而 H3 组蛋白家族 3A (Hist3A) 和核糖体蛋白 S18 (RpS18) 是最不稳定的基因。NormFinder 工具显示，Rpl4 是最稳定的基因，而 Elf1a 在所有测试条件下的排名均居中。BestKeeper 工具显示，Rpl4 和环磷酸腺苷 (CycA) 分别是更稳定和不太稳定的基因。这些数据共同表明，在 R. microplus 胚胎发生过程中，Rpl4、Elf1a 和 GAPDH 是 qPCR 标准化的合适内参。这些基因在本研究中使用的各种分析方法中均被一致鉴定为最稳定的基因。因此，本研究中的发现为研究 R. microplus 胚胎发生过程中的基因表达提供了有价值的信息。
