|Public on Mar 11, 2006
|condition name: Sox17-MO
Developmental Stage: embryonic stage 11
|Embryo manipulations and microinjections were preformed using standard procedures and embryos were staged according to the normal table of development for Xenopus laevis (Nieuwkoop and Faber, 1994). Two-cell stage embryos were vegetally injected with the following doses of antisense morpholino oligos or synthetic RNA: a combination of antisense morpholino oligos to Sox17a1 + Sox17a2 + Sox17b (20ng each) (Clements et al., 2003), Mixer antisense morpholino oligo (40ng) (Kofron et al., 2004), Cerberus-short RNA (1ng). For each biological sample for profiling, ~20 sibling embryos from a single mating or ~50 micro-dissected explants from sibling embryos were combined for individual RNA sample preparation.
|Total RNA was extracted using Trizol (Invitrogen) and purified on RNAeasy columns (Qiagen). Ten micrograms of total RNA was used for cDNA syntheses and labeled RNA probe which was hybridized to Affymetrix Xenopus Genechips by the Cincinnati Children's Research Foundation microarray core facility, using the standard Affymetrix protocol.
|Quality of total RNA is verified by the Agilent Bioanalyzer 2100 (Hewelett Packard) using the RNA 6000 Nano Assay according to manufactures recommendations. Then 10 micrograms of total RNA is used to make cDNA, from which botinylated cRNA probe was prepared using the standard Genechip Expression 3’ Amplification One-Cycle Target Labeling Kit (Affymetrix).
|20 micrograms of labeled cRNA in a volume of 200 microliters was hybridized to the Affymetrix Xenopus Genecip in a standard Affymetrix Probe Array Cartridge at 45 degrees Celsius, using the fluidics protocol EukGE-WS2v4_450 of a Fluidics Station 450 (Affymetrix).
|The image file was captured on an Affymetrix GeneChip Scanner 3000 and initially processed with Genechip Operating Software 1v4 (Affymetrix).
|Sample Number: 30
Experimental condition: Sox17 depleted by antisense morpholino oligos
|Standard Affymetrix internal control genes were used to check the quality of the assay quality by the signals of the 3' probe set to the 5' probe set of the internal control genes, GAPDH and B-actin, with acceptable 3' to 5' ratios between 1-3. Prokaryotic Spike controls were used to determine the hybridization of target RNA to the array occurred properly.
Each array was normalized the same. Global scaling is used adjusting the average intensity or signal value of each probe array to the same Target Intensity value (TGT) of 1500. Genechip Operating Software 1v4 (Affymetrix) was then used to generate CEL files from each experiment, which were then imported into GeneSpring for all further data analysis.
GeneSpring 7.1 software (Silicon Genetics) was used for data normalization, clustering and filtering. Raw CEL file data from all the samples was pre-normalized using RMA (Robust Multichip Average). The average log intensity of the biological replicates was then normalized to the average log intensity of stage 11 whole embryo.
|Mar 10, 2006
|Last update date
|Mar 10, 2006
|Aaron M Zorn
|Cincinnati Children's Hospital Medical Center
|3333 Burnet Ave
|Global analysis of the transcriptional network controlling Xenopus endoderm formation