The influence of RNA integrity on the detection of honey bee viruses: molecular assessment of different sample storage methods

RNA quality has been considered to be one of the most critical components for the overall success of RNA-based assays. To ensure accuracy of virus diagnosis by the RT-PCR method, it is important to identify an optimal sample storage method that stabilizes RNA and protects RNA from the activities of RNase in intact samples. We conducted studies to evaluate the effects of seven different storage conditions on the integrity of RNA and the influence of RNA integrity on the detection of virus infections in honey bees. RNA was isolated from samples processed under one of six storage conditions: 1) bees stored at 4 degrees C; 2) bees stored at -20 degrees C; 3) bees stored at -80 degrees C; 4) sliced bees immersed in RNAlater at 4 degrees C; 5) crushed bee immersed in RNAlater at 4 degrees C; 6) intact bees immersed in RNAlater at 4 degrees C, or 7) bees immersed in 70% ethanol at room temperature. The results indicated that bee samples stored at -80 degrees C, -20 degrees C, cut in slices and then immersed in RNAlater at 4 degrees C, and crushed into a paste and then immersed in RNAlater at 4 degrees C provided successful RNA stabilization, suggesting any one of these four storage methods is the method of choice for storing bee samples intended for virus analysis. RNA extracted from bee samples stored at 4 degrees C or whole bees immersed in RNAlater at 4 degrees C was partially degraded one week post treatment, suggesting that a temperature of 4 degrees C could not prevent RNA from activities of RNase completely and that the size of tissue is critical for successful stabilization of samples immersed in RNAlater. 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 relevant to other researchers and to apiary inspectors involved in epidemiological surveillance of bee viral infections.