Abstract

Quantitative real-time PCR (qRT-PCR) is a powerful technique to quantify gene expression. To standardize gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to consistently expressed housekeeping genes (HKGs) is required. In this study, ten candidate HKGs including elongation factor 1 α (EF1A), ribosomal protein L11 (RPL11), ribosomal protein L14 (RPL14), ribosomal protein S8 (RPS8), ribosomal protein S23 (RPS23), NADH-ubiquinone oxidoreductase (NADH), vacuolar-type H+-ATPase (ATPase), heat shock protein 70 (HSP70), 18S ribosomal RNA (18S), and 12S ribosomal RNA (12S) from the cowpea aphid, Aphis craccivora Koch were selected. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method were employed to evaluate the expression profiles of these HKGs as endogenous controls across different developmental stages and temperature regimes. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidate HKGs, RPS8, RPL14, and RPL11 were the three most stable HKGs across different developmental stages and temperature conditions. This study is the first step to establish a standardized qRT-PCR analysis in A. craccivora following the MIQE guideline. Results from this study lay a foundation for the genomics and functional genomics research in this sap-sucking insect pest with substantial economic impact.

Document Type

Article

Publication Date

6-19-2015

Notes/Citation Information

Published in PLOS One, v. 10, no. 6, article e0130593, p. 1-10.

© 2015 Yang et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Digital Object Identifier (DOI)

http://dx.doi.org/10.1371/journal.pone.0130593

Funding Information

This research was supported by a start-up fund from the University of Kentucky to XGZ, a grant from USDA BRAG grant (Award Agreement No.: 3048108827) to XGZ, and a Special Fund for Agroscience Research in the Public Interest (Award Agreement No.: 201303028) to YL. The granting agencies have no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

S1_Fig.tif (554 kB)
S1 Fig. The agrose gel electrophoresis of the ten candidate reference genes.

S2_Fig.tif (1054 kB)
S2 Fig. Melting curves of ten candidate reference genes in Aphis craccivora.

S1_Table.docx (11 kB)
S1 Table. The mean and standard deviation (SD) of the Ct value for each candidate reference gene.

S2_Table.docx (14 kB)
S2 Table. Summary of mean and SD values of gene pairwise comparison using the ΔCt method across different temperature.

S3_Table.docx (16 kB)
S3 Table. Summary of mean and SD values of gene pairwise comparison using the ΔCt method under the developmental stage.

S4_Table.docx (13 kB)
S4 Table. Ranking of the candidate reference genes by BestKeeper.

S5_Table.docx (14 kB)
S5 Table. Ranking of the candidate reference genes by BestKeeper.

journal.pone.0130593.g001.PNG (34 kB)
Fig 1 (PNG). Expression profiles of candidate housekeeping genes in Aphis craccivora.

journal.pone.0130593.g001.ppt (44 kB)
Fig 1 (PPT). Expression profiles of candidate housekeeping genes in Aphis craccivora.

journal.pone.0130593.g001.TIF (282 kB)
Fig 1 (TIFF). Expression profiles of candidate housekeeping genes in Aphis craccivora.

journal.pone.0130593.g002.PNG (29 kB)
Fig 2 (PNG). Pairwise variation (V) analysis of the candidate reference genes based on geNorm.

journal.pone.0130593.g002.ppt (35 kB)
Fig 2 (PPT). Pairwise variation (V) analysis of the candidate reference genes based on geNorm.

journal.pone.0130593.g002.TIF (270 kB)
Fig 2 (TIFF). Pairwise variation (V) analysis of the candidate reference genes based on geNorm.

journal.pone.0130593.t001.PNG (92 kB)
Table 1 (PNG). Summary of the ten housekeeping genes tested in this study.

journal.pone.0130593.t001.ppt (127 kB)
Table 1 (PPT). Summary of the ten housekeeping genes tested in this study.

journal.pone.0130593.t001.TIF (459 kB)
Table 1 (TIFF). Summary of the ten housekeeping genes tested in this study.

journal.pone.0130593.t002.PNG (69 kB)
Table 2 (PNG). A summary of ranking for reference gene candidates using five different statistical approaches.

journal.pone.0130593.t002.ppt (95 kB)
Table 2 (PPT). A summary of ranking for reference gene candidates using five different statistical approaches.

journal.pone.0130593.t002.TIF (330 kB)
Table 2 (TIFF). A summary of ranking for reference gene candidates using five different statistical approaches.

journal.pone.0130593.t003.PNG (70 kB)
Table 3 (PNG). A summary of ranking for reference gene candidates using five different statistical approaches.

journal.pone.0130593.t003.ppt (95 kB)
Table 3 (PPT). A summary of ranking for reference gene candidates using five different statistical approaches.

journal.pone.0130593.t003.TIF (329 kB)
Table 3 (TIFF). A summary of ranking for reference gene candidates using five different statistical approaches.

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