Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber)

Saumik Basu; Adriano E. Pereira; Daniele H. Pinheiro; Haichuan Wang; Arnubio Valencia-Jiménez; Blair D. Siegfried; Joe Louis; Xuguo Joe Zhou; Ana Maria Vélez

doi:10.1038/s41598-019-47020-y

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber)

Saumik Basu, Adriano E. Pereira, Daniele H. Pinheiro, Haichuan Wang, Arnubio Valencia-Jiménez, Blair D. Siegfried, Joe Louis, Xuguo Joe Zhou, Ana Maria Vélez

Entomology

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20 Scopus citations

Abstract

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3^rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.

Original language	English
Article number	10703
Journal	Scientific Reports
Volume	9
Issue number	1
DOIs	https://doi.org/10.1038/s41598-019-47020-y
State	Published - Dec 1 2019

Bibliographical note

Publisher Copyright:
© 2019, The Author(s).

Funding

The authors would like to thank Dr. Thais B. Rodrigues for suggestions on the experimental design and to Molly Darlington for helping with sample collections. The work in Dr. Vélez Lab was supported by start-up research funds by the University of Nebraska-Lincoln. The work in Dr. Joe Louis lab was partially supported by the Nebraska Agricultural Experiment Station with funding from the Hatch Act (Accession # 1007272) through the USDA National Institute of Food and Agriculture and start-up research funds by University of Nebraska-Lincoln.

Funders	Funder number
National Institute of Food and Agriculture
University of Nebraska–Lincoln
Alabama Agricultural Experiment Station	1007272

ASJC Scopus subject areas

General

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abstract = "Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.",

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Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, Diabrotica undecimpunctata howardi (Barber)

Abstract

Bibliographical note

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