All worms for embryo microarray expression experiments were first grown to high density on NG agar plates with concentrated HB101 at 20°C. For empty vector control, synchronous L1 larvae were spotted onto RNAi plates (NG agar with 100ug/ml Carb and 1mM IPTG) with concentrated HT115 bacteria carrying an empty RNAi vector (L4440). Worms were grown for ~96 hours at 20°C before collecting embryos by bleaching. For smo-1 RNA and sdc-2 RNAii, synchronous L1 larvae were first grown on HB101 for 24 hours to reach the L2/L3 stage and then transferred to RNAi plates spotted with concentrated HT115 bacteria carrying an Ahringer feeding library bacteria plasmid expressing double stranded RNA to smo-1 (Kamath et al. 2003). Worms were grown for 72 hours at 20°C on RNAi before harvesting embryos by bleaching.
Extracted molecule
total RNA
Extraction protocol
Gravid adults were bleached and 100 µl aliquots of embryos were snap frozen in liquid nitrogen and stored at -80C. An aliquot was thawed at 65C for 10 minutes, resuspended in 1ml Trizol (Invitrogen, Carlsbad, CA), vortexed vigorously to resuspend, and incubated for 20 minutes at room temperature. The sample was then centrifuged for 15 minutes at 12,000xg at 4C. The aqueous phase was transferred to a new tube and 100 µl of BCP (1-bromo-3-chloropropane) (MRC, Cincinnati, OH) was added, the sample was mixed by vortexing, incubated for 15 minutes at room temperature, and centrifuged for 10 minutes at 12,000xg at 4C. The aqueous phase was transferred to a new tube and nucleic acid was precipitated by addition of 500 µl of isopropanol. The sample was incubated for 10 minutes at room temperature, centrifuged at 12,000xg for 8 minutes, washed in 70% ethanol and air dried for 5 minutes. Nucleic acid was resuspended in 100 µl nuclease-free water and RNA quantified with a UV spectrophotometer. One hundred micrograms of RNA was then treated with 3 µl RQ1-DNAse (Promega, Madison, WI) and RNA isolated with the RNAeasy kit as per the manufacturer's protocol (Qiagen, Valencia, CA). RNA was again quantified with a UV spectrophotometer and cDNA was prepared from 5 µg RNA using the Protoscript Kit (NEB, Ipswich, MA) and diluted 1:15 in water.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 8 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix)
Hybridization protocol
Following fragmentation, 15 micrograms of cRNA were hybridized for 16 hr at 45C on GeneChip C. ElegansGenome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix Scanner 30007G.
Description
sdc-2(y93,RNAi) mixed stage embryos
Data processing
Arrays were normalized by RMA (Robust Multichip Average) using Affymetrix Expression Console software (Irizarry et al. 2003). Data were analyzed by ArrayStar. Statistical analysis was performed using a moderated t-test with FDR multiple testing correction (Benjamini-Hochberg method). Irizarry, R.A., Hobbs, B., Collin, F., Beazer-Barclay, Y.D., Antonellis, K.J., Scherf, U., and Speed, T.P. 2003. Exploration, Normalization, and Summaries of High Density Oligonucleotide Array Probe Level Data. Biostatistics 4: 249-264.
