Honey bee sample preparation

We determined that analysis of five honey bees per sample was sufficient for our colony monitoring project. Arthropod pathogen microarray (APM) analysis of test samples revealed that combined analysis of 5 bees reproducibly detected most, if not all, of the pathogens detected from 10 or 15 independently analyzed bees from the same sample. In addition, we confirmed the consistency of APM results by performing multiple analyses of a single RNA sample. Based on our test results and practical sample handling considerations, we reasoned that repeated analysis of 5 bees from each colony over-time (115 bees per colony) was sufficient for this study.

Honey bee samples, 5 bees per colony each time-point, were homogenized in 1 mL 50% TRIzol Reagent (Sigma) and 50% phosphate buffered saline (PBS, UCSF Cell Culture) solution in a 2 mL micro-centrifuge tube containing one sterile zinc-coated steel ball bearing (5 mm) using a TissueLyzer II (Retsch), for 4 minutes at 30 Hz. RNA was isolated according to TRIzol Reagent (Invitrogen) manufacturer's instructions. In brief, TRIzol reagent honey bee homogenate was combined with 0.1 ml chloroform and mixed by vortexing for 5 seconds, samples were incubated at room temperature for 5 minutes, prior to centrifugation for 10 minutes at 13,200× g in a table top centrifuge. Next, 700 µL of the aqueous phase was transferred to a new microfuge tube containing 490 µL isopropanol. Following mixing, the samples were incubated at −20°C for 20 minutes and then either centrifuged (13,200× g for 15 min) or further purified utilizing Zymo-III RNA columns according to manufacture's instructions (Zymo). RNA was extracted from five bees collected from the colony entrance for each of the time-course samples.