Food source: 90C lac-deleted strain of E. coli used as a food source (obtained from the MRC Laboratory of Molecular Biology at Cambridge (UK))
Treatment protocol
Microwave exposure and dosimetry: One group of worms was exposed to the microwave field (CW; 1.8 GHz; 5-40 mW kg-1) for 2.5 h at 26°C in the modified silver-plated TEM cell described in Dawe et al [2006], whereas the other group was sham-exposed (no field) for the same length of time at the same temperature in an unmodified copper TEM cell. The temperature difference between exposed samples in the silver-plated cell and sham samples in the copper cell has previously been measured at 0.1°C [Dawe et al, 2006], thereby minimising thermal artefacts in this study. Both TLM (N. Vasic & D. Thomas, unpublished) and FDTD modelling (G. Bit-Babik & M. Swicord, unpublished) concur with actual field measurements (B. Loader & A. Gregory, unpublished) that the SAR experienced by worms crawling across the well floor in a microwave-exposed 24-well multiwell plate varies from 5 mW kg-1 in the central wells up to 40 mW kg-1 in the corner wells.
Growth protocol
Worm culture: N2 worms were cultured at 15°C on large 14 cm Petri dishes containing Nematode Growth Medium (NGM) agar overlaid with a lawn of food bacteria (E. coli strain P90C), as described previously [Sulston & Hodgkin, 1988; Dawe et al, 2006]. Worms were then synchronised by egg isolation using bleach [Suslton & Hodgkin, 1988] and the L1 offspring were filtered using 5um nylon filters [Mutwakil et al, 1997]. These synchronised cultures were grown up to the L4 stage before exposure. L4 worms were washed off the plates using K-medium (53 mM NaCl, 32 mM KCl) [Williams & Dusenbery, 1990], filtered using 5um nylon filters to remove excess bacteria and then dispensed carefully using a magnetic stirrer into two equal groups of 6-well plates destined for microwave or sham exposure, respectively. The sample volume in each well was always 1.0 ml, as previously used for dosimetry and temperature measurements.
Extracted molecule
total RNA
Extraction protocol
Aliquots of L4 worms were either sham-exposed (control; no field) in the copper TEM cell, or else exposed to 1GHz, 0.5W for 2.5hours at 26C in the silver-plated TEM cell. Post exposure, the worms were quickly removed from the 6-well dishes using glass pipettes, pelleted by centrifugation (3000 x g for 3 min) and then dropped in small concentrated pellets (again using a glass pipette) into liquid nitrogen. These pellets were then crushed using a pre-cooled mortar and pestle (-80°C) in the presence of 2ml of Trizol (Invitrogen). The worm-Trizol slurry was transferred by spatula into a 50 ml tube, left to defrost at room temperature with regular agitation, and then transferred to 2 x 1.5 ml microcentrifuge tubes. A standard Trizol RNA extraction was then performed according to the manufacturer's instructions. All microcentrifuge tubes, the spatula and the mortar and pestle were autoclaved and pretreated with RNaseZap (Ambion, Huntingtdon, UK) and diethyl pyrocarbonate-treated water prior to use. RNA samples from three exposure runs (performed on different days) were combined for hybridization onto microarrays. Thus the results below derive from 5 sets of sham gene-arrays pooled from 15 sham exposures, compared against 5 sets of exposed gene-arrays pooled from 15 microwave exposures. RNA was transported on dry ice and storage was at -80°C.
Label
biotin
Label protocol
RNA quality was analysed with the Agilent 2100 Bioanalyser (Agilent Technologies, Geneva, Switzerland) using the RNA 6000 nano kit. All 10 RNA samples were of sufficient quality for gene array analysis. Approximately 5 µg of total RNA from each sample was used to produce cDNA using the GeneChip® One-cycle cDNA synthesis kit (Affymetrix, USA), as per manufacturer's instructions. Double stranded cDNA products were purified using the GeneChip® Sample Cleanup Module (Affymetrix, Santa Clara, USA). The synthesised cDNAs were in-vitro transcribed by T7 RNA polymerase using biotinylated nucleotides to generate biotinylated complementary RNAs (cRNAs) using the GeneChip® HT IVT labeling kit (Affymetrix, Santa Clara, USA), according to the manufacturer's instructions. The cRNAs were purified using the GeneChip® Sample Cleanup Module (Affymetrix, Santa Clara, USA). The cRNAs were then randomly fragmented at 94°C for 35 minutes in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate to generate molecules of approximately 35 to 200bp. Affymetrix C. elegans Genome GeneChip® arrays were hybridised with 15 µg of fragmented labeled cRNA for 16 h at 45°C as described in the Affymetrix Technical Analysis Manual using the GeneChip® hybridization control kit and GeneChip® hybridisation, wash and stain kit (Affymetrix, Santa Clara, USA). GeneChip® arrays were stained with streptavidin-phycoerythrin solution and scanned with an Affymetrix G2500A GeneArray scanner. Following scanning, non-scaled RNA signal intensity (CEL) files were generated using GeneChip® operating software (GCOS; Affymetrix, Santa Clara, USA) and normalised data was generated with the GCOS software using the MAS 5 algorithm (Affymetrix microarray suite users guide).
Hybridization protocol
Biotin Labelled cRNA using Affymetrix Enzo Kit according to guidelines set out by Affymetrix, Santa Clara, CA.
Scan protocol
Scanned according to guidelines set out by Affymetrix, Santa Clara, CA.
Description
wild-type (N2) strain of C. elegans (originally obtained from the MRC Laboratory of Molecular Biology at Cambridge (UK))
Data processing
The non-scaled RNA CEL files, were loaded into GeneSpring analysis software (GeneSpring 7.3; Agilent Technologies, Geneva, Switzerland) using the Robust Multichip Average (RMA) pre-normalisation algorithm (Irizarry et al., 2003). Further normalisations were performed for each experiment using a three step process: (i) probe-sets with a signal value <0.01 were set to 0.01, (ii) per chip normalisation to the 50th percentile, (iii) each gene signal from the microwave treated sample was normalised to the corresponding sham sample and the sham samples normalised to themselves.
