genotype/variation: Gad2-IRES-Cre electroporated with Myc and dominant negative p53
Treatment protocol
Tumor cells were dissociated and purified by percoll-gradient before RNA extraction.
Growth protocol
Spontaneous SHH medulloblastomas [dka001-005, 009, 033 and 034] and [dka050-057] were isolated from [Nestin-Cre; Cdkn2c-/-; Trp53fl/fl] and [Cdkn2c-/-; Ptch1+/-] mice (Uziel et al.,2005 Genes Dev). Spontaneous WNT medulloblastomas [pgr003, 016 and 066] were dissected from [Blbp-Cre; Ctnnb1+/lox (ex3); Trp53fl/fl] mice (Gibson et al., 2010, Nature). Group3 medulloblastomas [dka013-16, 049 and 065-067] were generated by overexpressing Myc in P6 cerebellar granule neuron progenitors (GNPs) from [Cdkn2c-/-, Trp53-/-] mouse pups prior to orthotopic injection into immunocompromised nude mice (Kawauchi et al, 2012, Cancer Cell). Cell-lineage specific Group3 medulloblastomas [dka072-075] and [dka077-081] were generated as in Kawauchi and colleagues (2012) except GNPs were isolated from Cre-recombined postnatal day 6 [Atoh1-CreER;Trp53fl/-] or [Prom1-CreER; Trp53fl/-] mouse pups (tamoxifen administered on P0-1), respectively. In situ Group3 medulloblastomas were generated by electroporating plasmids containing Myc and dominant negative Trp53 flanked by loxP sites into the fourth ventricle of E13.5 mouse embryos of Blbp-Cre [dka081, 087, 089, 090, 091 and blm121], Gad2-IRES-Cre [blm128-130 and blm134] or Ptf1a-Cre [blm135-137] mice. FACS-sorted GFP-positive [dka040, 042, 044] GNPs were obtained from P6 [Cdkn2c-/-, Trp53-/-, Atoh1-GFP] mouse pups.
Extracted molecule
total RNA
Extraction protocol
Total RNAs were extracted with Trizol reagent (Invitrogen) according to a supplied RNA isolation protocol.
Label
Biotin (Affyexpress)
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix one-Cycle protocol). Some samples were amplified and labeled using the Affyexpress method. Three samples were amplified by both method to serve as basis for batch correction. Biotinylated cRNA were prepared according to the standard Affymetrix one-Cycle protocol). Some samples were amplified and labeled using the Affyexpress method. Three samples were amplified by both method to serve as basis for batch correction
Hybridization protocol
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse Genome 430 2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GeneChip Scanner 3000 7G
Data processing
The data were analyzed with Partek Genomics Suite version 6.5 using the RMA method batch correction for amplficiation assay followed using the three repeated samples assayed by both methods in STATA/SE 11. Data provided in the spreadsheet are the corrected data. The Median Absolute Deviation (MAD) was calculated for each probe and then ranked to identify the 1000 most varying genes within the dataset (Following samples not included in calculation: dka040, 042 and 044 as these samples are not tumors; dka012, dka016 and dka049 since these were duplicate samples used for correction). Spotfire was used for two-way unsupervised hierarchical clustering and principal component analyses. Initial unsupervised hierarchical analysis used dka012, dka016 and dka049 and found them to cluster with or near their duplicate, so these were not used in the remainder of the analyses.
In vivo electroporation-based conditional oncogenic activation implicates multiple distinct cellular origins for Group3 medulloblastoma
Data table header descriptions
ID_REF
VALUE
Log2 RMA signal followed by correction factor applied to all data was derived to remove protocol effect mean(log2 values) of dka012,19 & 49 vs mean(dka065,66 & 67) and applied to all chips defines as labelled corr.
