Sample preservation, transport and processing strategies for honeybee RNA extraction: Influence on RNA yield, quality, target quantification and data normalization

- RNA degraded rapidly for samples not stored at −80 °C or processed within 24 h.
- Internal reference genes decay at a different rate than the virus targets.
- An extra homogenization step improves RNA yield and chemical purity in bee samples.
- The order of importance for RNA integrity is time > temperature > preservatives.
- β-actin mRNA is unsuitable as an internal reference gene for bee samples sent alive.

Viral infections in managed honey bees are numerous, and most of them are caused by viruses with an RNA genome. Since RNA degrades rapidly, appropriate sample management and RNA extraction methods are imperative to get high quality RNA for downstream assays. This study evaluated the effect of various sampling-transport scenarios (combinations of temperature, RNA stabilizers, and duration) of transport on six RNA quality parameters; yield, purity, integrity, cDNA synthesis efficiency, target detection and quantification. The use of water and extraction buffer were also compared for a primary bee tissue homogenate prior to RNA extraction. The strategy least affected by time was preservation of samples at −80 °C. All other regimens turned out to be poor alternatives unless the samples were frozen or processed within 24 h. Chemical stabilizers have the greatest impact on RNA quality and adding an extra homogenization step (a QIAshredder™ homogenizer) to the extraction protocol significantly improves the RNA yield and chemical purity. This study confirms that RIN values (RNA Integrity Number), should be used cautiously with bee RNA. Using water for the primary homogenate has no negative effect on RNA quality as long as this step is no longer than 15 min.