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Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers
Guillermo A Maroniche1, Mónica Sagadín, Vanesa C Mongelli
1Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (IB-INTA), Las Cabañas y Los Reseros s/n, Hurlingham CP 1686, Buenos Aires, Argentina.
This study identifies the most stable reference genes for quantitative gene expression analysis in the Mal de Río Cuarto virus (MRCV)-infecting planthopper, Delphacodes kuscheli. Polyubiquitin C (UBI), 18S, and actin (ACT) are recommended for accurate viral transmission studies.
Area of Science:
- Entomology
- Molecular Biology
- Plant Pathology
Background:
- Planthoppers transmit plant viruses, impacting crops and requiring molecular studies for understanding transmission.
- Quantitative real-time RT-PCR (RT-qPCR) is essential for gene expression analysis but needs reliable reference genes for normalization.
- Identifying stable reference genes in virus-infected planthoppers is crucial for accurate molecular research.
Purpose of the Study:
- To isolate and validate reference gene sequences in Delphacodes kuscheli, a vector of Mal de Río Cuarto virus (MRCV).
- To assess the expression stability of commonly used reference genes in MRCV-infective and naïve planthoppers.
- To establish a reliable RT-qPCR platform for gene expression studies in this insect vector.
Main Methods:
- Isolation of partial sequences for actin (ACT), α1-tubulin (TUB), GAPDH, EF1A, RPS18, and UBI genes.
- Design of specific RT-qPCR primers for selected reference genes.
- Evaluation of gene expression stability using geNorm, Normfinder, and BestKeeper analysis in infected and uninfected planthoppers.
Main Results:
- Polyubiquitin C (UBI), 18S, and actin (ACT) demonstrated the highest expression stability in MRCV-infective planthoppers.
- α1-tubulin (TUB) and elongation factor 1 alpha (EF1A) exhibited the most significant expression variability.
- Elongation factor 1 alpha (EF1A) expression was found to be upregulated following MRCV infection.
Conclusions:
- A validated RT-qPCR platform for gene expression analysis in MRCV-infected Delphacodes kuscheli was developed.
- This study provides the first report on reference gene selection in virus-infected insects, setting a precedent for future research.
- The findings are critical for advancing studies on MRCV transmission and other virus-planthopper pathosystems.
