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Title: THE INFLUENCE OF RNA INTEGRITY ON THE DETECTION OF HONEY BEE VIRUSES: MOLECULAR ASSESSMENT OF DIFFERENT SAMPLE STORAGE METHODS.

Author
item Chen, Yanping - Judy
item Evans, Jay
item Hamilton, Michele
item Feldlaufer, Mark

Submitted to: Journal of Apicultural Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/6/2007
Publication Date: 6/1/2007
Citation: Chen, Y., Evans, J.D., Hamilton, M.C., Feldlaufer, M.F. 2007. The influence of RNA integrity on the detection of honey bee viruses: Molecular assessment of different sample storage methods. Journal of Apicultural Research. 46(2):81-87.

Interpretive Summary: Honey bee samples collected from the field are often required to be shipped to laboratory facilities for determination of virus infections. However, bee samples can easily be degraded by the ubiquitous presence of enzymes during the shipping process if storage conditions are not ideal. The degradation of bee samples can result in a failure to detect viruses in virus-infected bees. Therefore, storage conditions during the period between the collection of the samples and the processing of the samples are of particular importance for accurate diagnosis of bee virus infections. We studied the effects of six storage conditions on the detection of honey bee virus infections. Our results indicated that traditional frozen storage method is the method of choice for storing bee samples intended for virus analysis. This information can be used by other researchers and apiary inspectors to investigate honey bee colonies for virus infections.

Technical Abstract: RNA quality has been considered one of the most critical components for the overall success of RNA-based assays. To ensure accuracy of virus diagnosis by RT-PCR method, it is important to identify an optimal sample storage method that stabilizes RNA and prevents RNA from activities of RNase in intact samples. We studied the effects of six storage conditions on the integrity of RNA and the influence of RNA integrity on the detection of honey bee virus infections. RNA was isolated from samples processed under each of six storage conditions: 1) at 4oC; 2) at –20oC; 3) at –80oC; 4) immersed in RNAlater at 4oC; 5) immersed in self-made RNA stabilization solution at 4oC; or 6) immersed in 70% ethanol at room temperature. The results indicated that frozen samples yielded RNA of the highest quality and the highest quantity among all tested samples, suggesting that the traditional frozen storage method is the method of choice for storing bee samples intended for virus analysis. RNAlater and self-made RNA stabilization solution only provide protection to tissues that are cut in very thin slices so that every part of the tissue can have direct contact with the solution. Therefore, they didn’t provide successful RNA stabilization for the whole intact bees in our studies. RNA from samples stored at 4oC was partially degraded, suggesting that a temperature of 4oC can slow down the activities of RNAse and provide a certain level of protection to RNA. 70% ethanol caused quick and strong degradation of RNA and therefore bee samples that are stored in 70% ethanol are not the recommended starting material for virus analysis. The information obtained from this study is interesting to other researchers and apiary inspectors involved in epidemiological surveillance of bee viral infections.