Pairwise variation analysis to determine optimum number of reference genes for accurate normalization.

<p>A pairwise variation analysis (geNorm) globally for all samples (n = 12) as well as subset analysis on brood mite samples (n = 3), phoretic mites (n = 3), Low virus mites (n = 3) and High virus mites (n = 3). Each sample comprised 8 pooled mites. Pairwise variation is compared with 0.15, below which the inclusion of a subsequent reference gene is not necessary to ensure accurate normalization.</p

Stabilities of cycle threshold (Cq) values of ten candidate reference genes in <i>Varroa destructor</i>.

<p>The box plot with medians indicates the 25th and 75^th percentiles and the whiskers (error bars) indicate the 10^th and 90^th percentiles. Black dots represent outliers. Data for each gene from 3 replicates of each grouping (brood, phoretic, Low Virus and High Virus), each sample contained 8 varroa.</p

Electropherogram assessment of RNA integrity from pierced and unpierced <i>Varroa</i> mites collected into RNAlater after different storage conditions.

<p>Total RNA was extracted from either intact or pierced mites stored in RNAlater for 0, 3 and 10 days at room temperature. 50ng aliquots of RNA were run on the Agilent Bioanalyzer 2100 microfluidics gel analysis platform to generate electropherograms. Single 28S ribosomal peaks are seen in intact samples. Smaller peaks at 22, 26 and 28s are Agilent normalization markers and gel size ladders.</p

Validation of reference gene selection in a study of vitellogenin gene expression.

<p>Expression levels vitellogenin transcript was measured in brood phase mites (reproductive) relative to levels in phoretic mites (non-reproductive) using a combination of the most stable three (NADH + HSP90 + 18S), two (NADH + HSP90) and single (NADH) reference genes, and the least stable reference genes (actin and α-tubulin). Data are Means ± SEM, n = 3.</p

Candidate reference genes selected for stability analysis.

<p>Candidate reference genes selected for stability analysis.</p

Ranking of candidate reference genes.

<p>Ranking of candidate reference genes.</p

A Toolbox for Quantitative Gene Expression in <i>Varroa destructor</i>: RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability

<div><p><i>Varroa destructor</i> is the major pest of <i>Apis mellifera</i> and contributes to the global honey bee health crisis threatening food security. Developing new control strategies to combat <i>Varroa</i> will require the application of molecular biology, including gene expression studies by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Both high quality RNA samples and suitable stable internal reference genes are required for accurate gene expression studies. In this study, ten candidate genes (succinate dehydrogenase (SDHA), NADH dehydrogenase (NADH), large ribsosmal subunit, TATA-binding protein, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA (18S), heat-shock protein 90 (HSP90), cyclophilin, α-tubulin, actin), were evaluated for their suitability as normalization genes using the geNorm, Normfinder, BestKeeper, and comparative ΔCq algorithims. Our study proposes the use of no more than two of the four most stable reference genes (NADH, 18S, SDHA and HSP90) in <i>Varroa</i> gene expression studies. These four genes remain stable in phoretic and reproductive stage <i>Varroa</i> and are unaffected by Deformed wing virus load. When used for determining changes in vitellogenin gene expression, the signal-to-noise ratio (SNR) for the relatively unstable genes actin and α-tubulin was much lower than for the stable gene combinations (NADH + HSP90 +18S; NADH + HSP90; or NADH). Using both electropherograms and RT-qPCR for short and long amplicons as quality controls, we demonstrate that high quality RNA can be recovered from <i>Varroa</i> up to 10 days later stored at ambient temperature if collected into RNAlater and provided the body is pierced. This protocol allows the exchange of <i>Varroa</i> samples between international collaborators and field sample collectors without requiring frozen collection or shipping. Our results make important contributions to gene expression studies in <i>Varroa</i> by proposing a validated sampling protocol to obtain high quality <i>Varroa</i> RNA and the validation of suitable reference genes for expression studies in this globally important pest.</p></div

DWV-A, DWV-B and Pan-DWV in <i>A</i>. <i>mellifera</i> from heavily Varroa-infested hives.

<p>The DWV-A, DWV-B and Pan-DWV titres in five individual <i>A</i>. <i>mellifera</i> from the University of Aberdeen apiary employing the single plasmid construct as the external standard. Data presented are DWV genome equivalents per individual (total extracted RNA) mean ± SE performed in triplicate, n = 5.</p

Standard curves and linear uncertainty plots for Pan-DWV, DWV-A and DWV-B assays.

<p>Standard curves and linearity uncertainty (<i>U</i>_LINi) plots for each primer pair using the single constructed plasmid external standard containing fragments for A.) Pan-DWV, B.) DWV-A and C.) DWV-B. Data for the standard curve represent mean ± SE performed in quadruple. Linear regression performance was determined by calculating the mean bias for each load, with bars representing the linearity uncertainty (<i>U</i>_LINi).</p

DWV-A, DWV-B and Pan-DWV levels in <i>A</i>. <i>mellifera</i> from hives with differing Varroa infestations.

<p>DWV-A, DWV-B and Pan-DWV titres in <i>A</i>. <i>mellifera</i> from three apiaries in Scotland, A) Heavily Varroa-infested colonies at the University of Aberdeen apiary (n = 9), B) Lowly Varroa-infested colonies at an Aberdeenshire apiary (n = 8), and C) Varroa-free colonies at the Beinn Eighe National Park (n = 8). Data presented are DWV genome equivalents per individual (total extracted RNA), mean ± SE performed in triplicate.</p