Doxycycline chow was replaced with regular chow to switch on oncogene expression
Growth protocol
Mice were kept on doxy diet to keep oncogenes switched off
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using mirVANA kit from Ambion. The miRNA-enriched fraction of small RNAs was purified from total RNA by polyacrylamide gel electrophoresis using Ambion’s flashPAGE™ kits, Ambion Inc., Austin, TX.
Label
biotin
Label protocol
The 3’ ends of the RNA molecules were tailed and biotin-labeled using the mirVana™ miRNA Labeling Kit (Ambion Inc., Austin, TX). The kit’s dNTP mixture in the tailing reaction was replaced with a proprietary nucleotide mixture containing biotin-modified nucleotides (PerkinElmer, Waltham, MA).
Hybridization protocol
Hybridization, washing, staining, imaging, and signal extraction were performed according to Affymetrix-recommended procedures, except that the 20X GeneChip Eukaryotic Hybridization Control cocktail was omitted from the hybridization.
Scan protocol
The fluorecence data is extracted at 1.4 micron pixel resolution using the Affymetrix 3000 7G scanner.
Description
A0007684: MYC over-expressor
Data processing
The signal processing implemented is a multi-step process involving probe specific signal detection calls, background estimate and correction, constant variance stabilization and either array scaling or global normalization. For each probe, an estimated background value is subtracted that is derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific analysis experiment were normalized together according to the variance stabilization method described by Huber et al. (Huber et al., 2002). Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-Test, with assumption of equal variance, was applied. One-way ANOVA was used. These tests define which probes are considered to be significantly differentially expressed, or significant, based on a default p-value of 0.05 and log2 difference > 1. The post-normalized data scale is reported as generalized log2 data.
