| Autor: |
Kim, Tae Kwon, Waldman, Jéssica, Ibanez-Carrasco, Freddy, Tirloni, Lucas, Waltero, Camila, Calixo, Christiano, Braz, Gloria R., Mulenga, Albert, Junior, Itabajara da Silva Vaz, Logullo, Carlos |
| Zdroj: |
Ticks and Tick-borne Diseases; 20230101, Issue: Preprints |
| Abstrakt: |
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 microplusis 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. microplushousekeeping 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 Rpl4was the most stable gene, while the ranking of Elf1awas intermediate in all tested conditions. The BestKeeper tool showed that Rpl4and cyclophilin A(CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDHare suitable internal controls for normalizing qPCR during R. microplusembryogenesis. 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. |
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