Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring mRNA expression. To facilitate gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to stable housekeeping genes is mandatory. In this study, ten housekeeping genes, including beta-actin (Actin), elongation factor 1 α (EF1A), glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L13 (RPL13), ribosomal protein 49 (RP49), α-tubulin (Tubulin), vacuolar-type H+-ATPase (v-ATPase), succinate dehydrogenase subunit A (SDHA) , 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S) from the two-spotted spider mite, Tetranychus urticae, were selected as the candidate reference genes. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the performance of these candidates as endogenous controls across different developmental stages. In addition, RefFinder, which integrates the above-mentioned software tools, provided the overall ranking of the stability/suitability of these candidate reference genes. Among them, PRL13 and v-ATPase were the two most stable housekeeping genes across different developmental stages. This work is the first step toward establishing a standardized qRT-PCR analysis in T. urticae following the MIQE guideline. With the recent release of the T. urticae genome, results from this study provide a critical piece for the subsequent genomics and functional genomics research in this emerging model system.

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Published in PLOS One, v. 10, no. 3, article e0120833, p. 1-12.

© 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

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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. These agencies had no role in study design, data collection/analysis, manuscript preparation, or the decision to publish.

journal.pone.0120833.s001.TIFF (205 kB)
S1 Fig. The agrose gel profile of the ten candidate reference genes.

journal.pone.0120833.s002.TIF (2155 kB)
S2 Fig. Melting curve of the ten candidate reference genes.

journal.pone.0120833.s003.DOCX (11 kB)
S1 Table. The mean and standard deviation (SD) of the <em>C</em><sub>t</sub> values of the ten candidate reference gene.

journal.pone.0120833.s004.DOCX (14 kB)
S2 Table. Pairwise comparison of candidate reference genes.

journal.pone.0120833.s005.DOCX (13 kB)
S3 Table. Ranking of the candidate reference genes based on their crossing point (CP) values by BestKeeper.

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