7 protocols
AccessionType
image_acquisition
Honey bee gene expression microarray analysis

Microarrays were scanned using an Axon 4B scanner and Gene PixPro software (Molecular Devices) for gridding and creation of .gpr files for further analysis of the differentially expressed genes (DEGs) using Bioconductor’s Linear Models for Microarray Analysis (LIMMA) (Smyth, 2004) implemented in the R/Bioconductor platform (R-code provided) (Gentleman et al., 2004).


(Parameters: Scanning hardware = Axon- GenePix4000B, Scanning software = GenePix Pro [Axon Instruments])
nucleic_acid_extraction
Honey bee RNA was isolated from whole individual bees using TRizol reagent (900 ?l) (Invitrogen) according to manufacturer’s instructions. In brief, TRIzol reagent and honey bee homogenate was combined with 0.2 ml chloroform and mixed by vortexing for 5 seconds, samples were incubated at room temperature for 5 minutes, prior to centrifugation for 12 minutes at 13,200 x g in a table top centrifuge. Next, 700?L of the aqueous phase was transferred to a new microfuge tube containing 700 ?L ethanol. Following mixing, RNA was further purified using Qiagen RNAeasy columns, including on column DNase Treatment (Qiagen). RNA quality was assessed using an Agilent 2100 Bioanalyzer and Nanodrop spectrophotometer (ThermoScientific).
(Parameters: Extracted product = total_RNA, Amplification = none)
labeling
cDNA synthesis reactions were performed with SuperScriptIII (Invitrogen). In brief, RNA from five individual bees per treatment group (10 ?g), oligo(dT)20 primer (10 ?g) and random hexamer (500 ng) were combined in a 20 ?L reaction volume, incubated at 65°C (5 min), and cooled on ice (1 min) and subsequently combined with 20 ?L of 2X First-Strand Buffer containing SSIII (400 U), dNTPs (0.5 mM each dA/G/CTP; 0.2 mM dTTP; 0.3 mM aa-dUTP), DTT (5 mM) and RNaseOUT (80 U). Reverse transcription reactions were incubated for 12 hours at 42°C followed by inactivation of the reaction (70°C, 15 min). After cDNA synthesis, the RNA was hydrolyzed by NaOH (12 ?L of 1N) and EDTA (12 ?L of 0.5 M pH8.0) treatment (65°C 15 min), neutralized by addition of HEPES pH 7 (60 ?L). amino-allyl cDNA was purified using MinElute columns (Qiagen). aa-cDNA (1750 ng) from each individual bee was labeled with Cy5 (sample) and reference RNA was labeled with Cy3. Dye labeling reactions [aa-cDNA (1750 ng) +16 nmoles dye in 20 ?L buffer (0.1 NaOH pH 9) proceeded for 2 hours at RT in the dark, followed by MinElute column clean up and quantification. Hybridization A “reference RNA” strategy was utilized for comparative analysis individual honey bee gene expression (5 individuals per group, hybridized independently). 70 ?L of sample (containing Cy3-labeled sample and Cy3-labled reference; each normalized to 60 pmol dye) in hybridization buffer (25% formamide, 5x SSC, 0.1% SDS) was loaded under each lifterslip and arrays were hybridized at 42°C for 18 hours (Alaux et al., 2009). Post-hybridization washes were performed following removal of each lifter slip in wash #1 solution (2X SSC, 0.2% SDS), wash #1 for 10 minutes at 42°C; wash #2 (2X SSC, 0.2% SDS) for 10 minutes at 42°C; wash #3 (1X SSC) for 10 minutes; wash #4 (0.1X SSC) for 10 minutes. Slides were spun dry in an ozone-free hood.
(Parameters: Amount of nucleic acid labeled = 10, Amplification = none, Mass unit = Micro gram)
specified_biomaterial_action
Flenniken 2012; Honey bee gene expression microarray treatment groups:

To minimize variability between samples all arrayed bees were obtained from a single brood comb from a naturally mated queen, therefore all the bees were age-matched half-sisters. The bees selected for microarray analysis of virus (Sindbis-eGFP) co-injected with either specific-dsRNA or non-specific dsRNA exhibited the reduced virus phenotype that was seen in the majority of the bees assayed. The five representative bees from each condition (v, v+vs-dsRNA, v+ns-dsRNA, dsRNA, and mock) selected for microarray analysis were free of pre-existing conditions (assessed by APM analysis) (Runckel, Flenniken et al., 2011).
hybridization
Hybridization A “reference RNA” strategy was utilized for comparative analysis individual honey bee gene expression (5 individuals per group, hybridized independently). 70 ?L of sample (containing Cy3-labeled sample and Cy3-labled reference; each normalized to 60 pmol dye) in hybridization buffer (25% formamide, 5x SSC, 0.1% SDS) was loaded under each lifterslip and arrays were hybridized at 42°C for 18 hours (Alaux et al., 2009). Post-hybridization washes were performed following removal of each lifter slip in wash #1 solution (2X SSC, 0.2% SDS), wash #1 for 10 minutes at 42°C; wash #2 (2X SSC, 0.2% SDS) for 10 minutes at 42°C; wash #3 (1X SSC) for 10 minutes; wash #4 (0.1X SSC) for 10 minutes. Slides were spun dry in an ozone-free hood.
(Parameters: Chamber type = OTHER: DeRisiLabManufactured, Quantity of label target used = 750, Mass unit = Nano gram, time = 18, Tiny time unit = hours, Volume = 70, Volume unit = Micro litre, temperature = 42)
grow
Honey Bees (Apis mellifera) are not well adapted to the laboratory setting therefore for our experiments we used newly emerged honey bees (~24 hours old). Frames of emerging bees were taken from colonies in San Francisco, CA and housed in the laboratory. Young female worker bees (all half sisters) were collected daily and housed in modified deli-containers (similar to those described by Evans et al. 2009 (Evans et al., 2009)) at room temperature for the duration of the experiment. Water (provided on a sponge) and sugar (cube) was provided and replenished daily throughout the duration of the experiment. To minimize variability between samples all arrayed bees were obtained from a single brood comb from a naturally mated queen, therefore all the bees were age-matched half-sisters.
pool
To facilitate gene expression comparisons between multiple treatment groups we utilized a reference-design strategy in which each Cy5-labeled experimental sample was hybridized with a standardized Cy3-labeled reference sample. A complex RNA mixture representing hundreds of bees of various ages exposed to difference treatment groups, served as the reference RNA sample.