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Stable internal reference genes for quantitative RT-PCR analyses in Rhipicephalus microplus during embryogenesis.

Tae Kwon Kim1, Jéssica Waldman2, Freddy Ibanez-Carrasco3

  • 1Department 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.

Ticks and Tick-Borne Diseases
|September 14, 2023
PubMed
Summary

Identifying stable reference genes is crucial for accurate gene expression studies in the cattle tick Rhipicephalus microplus. This study found ribosomal protein L4 (Rpl4) and elongation factor 1α (Elf1a) to be the most reliable internal controls during embryogenesis.

Keywords:
QRT-PCRReference geneTick

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Area of Science:

  • Molecular Biology
  • Genomics
  • Parasitology

Background:

  • Accurate gene expression analysis relies on stable internal reference genes for normalization.
  • Constitutive gene expression can be complex and vary across different conditions, necessitating validation.
  • The cattle tick Rhipicephalus microplus is a significant pathogen vector, making its developmental gene expression critical to study.

Purpose of the Study:

  • To identify and evaluate the stability of housekeeping genes in Rhipicephalus microplus during embryogenesis.
  • To determine the most suitable reference genes for normalizing quantitative PCR (qPCR) data in tick embryogenesis research.

Main Methods:

  • Selection of candidate reference genes based on previous studies.
  • Screening of selected genes using quantitative PCR (qPCR) across different embryogenesis time points.
  • Analysis of gene expression stability using GeNorm, NormFinder, and BestKeeper tools.

Main Results:

  • Reference gene stability varied significantly during Rhipicephalus microplus embryogenesis.
  • Elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were identified as the most stable genes by GeNorm.
  • Rpl4 was consistently ranked as highly stable across multiple analysis tools, with Elf1a and GAPDH also showing suitability.

Conclusions:

  • Ribosomal protein L4 (Rpl4), elongation factor 1α (Elf1a), and GAPDH are recommended as reliable internal controls for qPCR normalization during Rhipicephalus microplus embryogenesis.
  • Validated reference genes are essential for accurate interpretation of gene expression data in tick developmental studies.
  • This research provides critical tools for advancing Rhipicephalus microplus embryogenesis research.