N2 wild type, DR subclone of CB original (Tcl pattern I) L4 larvae
Treatment protocol
L1 larvae (2.5 x 105) were suspended in T-75 flasks containing 30 mL CeHR medium and grown until 90% of the population had developed to the mid-vulval L4 larval stage. The worms were dosed with mefloquine (Ash Stevens, Inc., Detroit, MI), dichlorvos, or fenamiphos (Chem Service, Inc., West Chester, PA) for 8 h or allowed to develop as a control. Each exposure was repeated three times. Worms were harvested by centrifugation (800 x g for 3 min at 4° C), and the supernatant was aspirated. The pellets for were suspended in the residual liquid, flash frozen by drop-wise addition to liquid nitrogen, and stored at -80° C.
Growth protocol
C. elegans were maintained in synchronized cultures grown in CeHR medium. All cultures were grown at 22.5° C with shaking at 70 rpm. Typically, 5 x 105 L1 larvae were used to inoculate 40 mL of medium in a T-75 flask. Cultures were harvested every generation to maintain synchronization. CeHR medium is a sterile, defined medium, supplemented with 20% (v/v) ultrapasteurized organic, fat-free milk for the axenic propagation of C. elegans. A detailed description of the preparation of the medium is available from the USACEHR on request.
Extracted molecule
polyA RNA
Extraction protocol
Frozen worm droplets were pulverized in liquid N2 using a pre-chilled mortar and pestle. The pulverized worms were transferred to 6 mL Trizol (Invitrogen, Carlsbad, CA) and homogenized in a dounce homogenizer. RNA was purified according to the manufacturer’s protocol and precipitated with isopropyl alcohol. After centrifugation, the RNA pellet was dried, dissolved in water, and subjected to an additional round of purification using the RNEasy Maxi Kit (Qiagen, Valencia, CA) according to the manufacturer’s directions. The quality and yield of the preparation was assessed throughout processing and labeling using a 2100 Bioanalyzer (Agilent, Santa Clara, CA), and when necessary, the mass yield was confirmed using an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Poly(A)+ RNA was isolated from the total RNA using OligoTex (Qiagen) essentially as described by the manufacturer. Two micrograms of poly(A)+ RNA (adjusted for rRNA contamination) was used as the template for cDNA synthesis using the SuperScript Choice kit (Invitrogen) per the manufacturer’s recommendations except that (1) a high pressure liquid chromatography (HPLC)-purified T24T7 promoter primer (Integrated DNA Technologies, Coralville, IA) was used to initiate first strand synthesis; (2) the second strand synthesis was not terminated using EDTA since we found that EDTA carryover interfered with subsequent enzymatic manipulations; and (3) PelletPaint (Novagen, Madison, WI) was used in place of glycogen for precipitation.
Label
biotin
Label protocol
Biotin labeled cRNA was synthesized from the T7 promoter incorporated in the cDNA using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY) per the manufacturer’s recommendations; approximately 1 µg of cDNA was used for synthesis. cRNA was purified from unincorporated nucleotides and other reaction components using the RNEasy Mini kit (Qiagen).
Hybridization protocol
cRNA samples fragmented according Affymetrix recommendations were hybridized to C. elegans whole genome GeneChips (Affymetrix, Santa Clara, CA) at the Walter Reed Army Institute of Research Vaccine Genomics Laboratory, Rockville, MD using Affymetrix instrumentation and with hybridization and washing parameters provided by the manufacturer.
Scan protocol
The C. elegans whole genome GeneChips (Affymetrix, Santa Clara, CA) were scanned at the Walter Reed Army Institute of Research Vaccine Genomics Laboratory, Rockville, MD using Affymetrix instrumentation with scanning parameters provided by the manufacturer.
Description
nominal concentration 200 mg/L
Data processing
Microarray data was processed using the robust multi-array averaging method (RMA) as implemented in Partek Pro Genomics Suite v. 6.3. To verify inter-replicate reproducibility, replicate samples were subjected to pairwise correlation analysis of all probe sets. For the vast majority of replicate pairs, the R2 value was greater than or equal to 0.95, and no replicates were included with R2 < 0.92. A Present, Absent, or Marginal call for each probe set was determined using the R statistical package and the Bioconductor implementation of the Affymetrix MAS 5.0 algorithm (affy package 1.12.2).
