Abstract
Stored potato tubers are susceptible to pathogens, such as Potato virus Y, and studies of host/pathogen interactions on a gene transcription level can provide insight into the disease development. A method for studying individual gene expression is reverse trancription quantitative polymerase chain reaction (RT-qPCR) that relies on utilization of reference genes. To select appropriate reference genes with stable expression in our experimental setting, we screened the genome-wide microarray expression data for suitable candidate reference genes rather than to use generally recognised constitutively expressed housekeeping genes as reference genes. Four highly expressed genes with stable expression across several comparisons were selected based on microarray data. Stable expression of these candidate reference genes (Nicalin, Eukaryotic translation initiation factor 5A, Universal stress protein and Katanin p60 ATPase-containing subunit) was confirmed using RT-qPCR. Our candidate reference genes were more suitable than housekeeping genes often used as reference genes. Additionally, microarray expression data was evaluated for eight previously reported reference genes.
Resumen
Los tubérculos de papa almacenados son susceptibles a patógenos, como el virus Y de la papa, y estudios de la interacción hospedero/patógeno a nivel de la transcripción de un gen pueden proporcionar una visión al interior del desarrollo de la enfermedad. Un método para estudiar la expresión de un gen individual es una transcripción inversa cuantitativa de la reacción en cadena de la polimerasa (RT-qPCR) que confía en la utilización de genes de referencia. Para seleccionar los genes de referencia apropiados con expresión estable en nuestra configuración experimental, revisamos los datos de expresión de amplitud del genoma de microarreglos para genes candidatos de referencia deseables en vez de usar genes básicos generalmente reconocidos expresados constitutivamente como genes de referencia. Se seleccionaron cuatro genes altamente expresados estables a lo largo de varias comparaciones con base en datos de microarreglos. Se confirmó la expresión estable de estos genes candidatos de referencia (Nicalin, factor de iniciación 5A de translación eucariótica, Proteína universal de agobio y subunidad con Katanin p60 ATPasa) usando RT-qPCR. Nuestros genes candidatos de referencia fueron mas apropiados que los genes básicos a menudo usados como genes de referencia.
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Abbreviations
- 18S rRNA:
-
18S ribosomal RNA
- adj.p:
-
adjusted p value
- APRT:
-
Adenine phosphoribosyl transferase
- CT :
-
threshold cycle
- CYP:
-
Cyclophilin
- dCT :
-
delta threshold cycle
- EF1-α:
-
Elongation factor 1-α
- EIF5A:
-
Eukaryotic translation initiation factor 5A
- Hsp20.2:
-
Heat shock protein 20
- KATN:
-
Katanin
- L2:
-
Cytoplasmic ribosomal protein L2
- lfc:
-
logarithmic fold change, log2-fold change
- NCLN:
-
Nicalin
- PVY:
-
Potato virus Y
- PVYNTN :
-
strain of Potato virus Y that induces potato tuber necrotic ringspot disease
- RT-qPCR:
-
reverse trancription quantitative polymerase chain reaction
- SD:
-
standard deviation
- USP:
-
Universal stress protein
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Acknowledgments
This study was financially supported by the Slovenian Research Agency (project L4-2400-0401, programme P4-0072).
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Conceptualization: Barbara Gerič Stare; Methodology: Barbara Gerič Stare, Aleš Sedlar; Formal analysis and investigation: Barbara Gerič Stare, Aleš Sedlar; Writing - original draft preparation: Aleš Sedlar; Writing - review and editing: Barbara Gerič Stare, Vladimir Meglič; Funding acquisition: Vladimir Meglič.
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Aleš Sedlar is no longer employed here.
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Gerič Stare, B., Sedlar, A. & Meglič, V. Microarray-Based Uncovering of Reference Genes for Quantitative Real-Time PCR in Potato Tuber Infected with PVY. Am. J. Potato Res. 98, 202–209 (2021). https://doi.org/10.1007/s12230-021-09832-5
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DOI: https://doi.org/10.1007/s12230-021-09832-5