1.2.8. Dead bee samples

Many bee disease experiments involve bee death as a parameter. Dead bee samples from such experiments are, of course, valid material for analysis. They should be treated like freshly killed material and frozen as soon as possible to minimize the effects of decay on RNA integrity, using the collection methods appropriate for the sample type, as given above. Dead bee traps attached to hives are suitable for collecting such bees and should be emptied daily to minimize the effects of decomposition.

Passive surveys also involve dead bee samples, in this case those sent in by beekeepers for post-mortem analysis of the cause of colony death. These bees will have been dead long enough for decomposition and drying to have severely affected the integrity of the RNA, including viral RNAs. Such degradation can severely affect the accuracy and reliability of detecting and quantifying individual RNAs (Bustin and Nolan, 2004; Fleige and Pfaffl, 2006; Becker et al., 2010). This means that only positive results from such samples are informative, since negative results can be either due to the absence of virus or the degradation of the RNA.

It is possible to adjust for differential RNA degradation in the different samples with quantitative RT-qPCR techniques, by using host internal reference gene levels for normalizing the virus titers (Dainat et al., 2011) and setting the threshold for template detection with the most degraded sample, so that all samples are evaluated by the same degradation criteria. How to determine the detection thresholds using RT-qPCR assays is covered in section 4.4.