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Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti

Najat Dzaki1, Karima N Ramli1, Azali Azlan1

  • 1School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.

Scientific Reports
|March 17, 2017
PubMed

Insights

Accurate gene expression studies in the disease-vector mosquito Aedes aegypti require validated reference genes. This study identified actin (ACT) and ribosomal protein S17 (RPS17) as the most stable genes for normalization in Ae. aegypti research.

Area of Science:

  • Vector-borne disease research
  • Molecular biology
  • Genomics

Background:

  • Aedes aegypti mosquitoes transmit critical viruses like Dengue, Chikungunya, and Zika.
  • Quantitative real-time PCR (qPCR) is vital for studying gene expression in Ae. aegypti.
  • A lack of validated reference genes hinders accurate gene expression normalization in Ae. aegypti.

Purpose of the Study:

  • To identify and validate stable reference genes for accurate gene expression normalization in Ae. aegypti.
  • To assess the suitability of seven commonly used reference genes for qPCR studies in Ae. aegypti.

Main Methods:

  • Investigated expression stability of seven reference genes (ACT, eEF1α, α-tubulin, RPL8, RPL32, RPS17, GAPDH) across 16 developmental stages and cell culture.
  • Utilized three algorithms (BestKeeper, geNorm, NormFinder) to analyze qPCR data and infer gene stability.
  • Determined consensus rankings based on stability values from the algorithms.

Main Results:

  • Actin (ACT) and ribosomal protein S17 (RPS17) demonstrated the highest expression stability.
  • A combination of ACT and RPS17 is proposed as the optimal normalization strategy for Ae. aegypti.
  • Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) exhibited the least stability and is not recommended.

Conclusions:

  • Validated reference genes are crucial for reliable qPCR-based gene expression studies in Ae. aegypti.
  • The ACT/RPS17 combination provides a dependable normalization method for Ae. aegypti research.
  • This study provides essential guidelines for selecting reference genes in vector research.

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