genotype: human VHL P81S transgene in Vhl-/- mouse ES cells injected into nude mouse host phenotype: apoptosis-resistant tissue: teratoma host strain: *nu/nu* mice (Taconic Labs, Hudson, NY, USA) donor strain: wild-type J1 strain Sv/129 ES cells
Treatment protocol
Female nude mice were injected subcutaneously in bilateral flanks with 1x10^6 cells of each VHL ES cell line. When tumors reached a maximum dimension of 1cm, mice were sacrificed by CO2, and tumors were excised and flash frozen in liquid nitrogen.
Growth protocol
Vhl-/- mouse embryonic stem (ES) cells expressing wildtype, P81S or R167Q human VHL gene cDNA were grown to 70% confluency, trypsinized and suspended in 100uL of sterile PBS prior to injection.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from frozen teratoma samples using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. RNA concentration and quality were assessed using ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively.
Label
biotin
Label protocol
Total RNA samples were processed following a standard one-cycle eukaryotic target preparation protocol from Affymetrix. Briefly, total RNA was first reverse-transcribed using T7-oligo(dT) promoter primer in the first-strand cDNA synthesis reaction. Following RNase H-mediated second-strand cDNA synthesis, the double stranded cDNA was purified and served as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling.
Hybridization protocol
Biotinylated cRNA targets were then purified, fragmented, and hybridized to GeneChip® array during the overnight incubation at 45 °C in a rotating hybridization oven.
Scan protocol
After hybridization, the arrays were stained with streptavidin-phycoerythrin in the GeneChip® Fluidics 450 station and then scanned using Affymetrix GeneChip® Scanner 3000 7G Plus Targeted Genotyping System with Autoloader (laser filter set at 570 nm; pixel size 2.5 μm). The array image data were acquired, and the fluorescent signal intensities were quantified using Affymetrix® GCOS v. 1.2 software with following settings of quantitation parameters: Alpha1 = 0.05, Alpha2 = 0.065, Tau = 0.015, Gamma1H = 0.0045, Gamma1L = 0.0045, Gamma2H = 0.006, Gamma2L = 0.006, Perturbation = 1.1, Target Intensity = 150.
Description
Gene expression data from VHL mutant ES cell teratoma
Data processing
Gene expression data (CEL files) were analyzed using Partek® Genomic Suite, version 6.5 (Partek Inc., St. Louis MO) by robust multi-array analysis (RMA). The sequences from which the array probe sets were derived from GenBank®, Ensembl, dbEST, and RefSeq. Gene expression data (CEL files) were analyzed using Partek® Genomic Suite, version 6.5 (Partek Inc., St. Louis MO). Gene expression data were normalized by robust multi-array analysis (RMA) and analysis of variance was performed using Partek® software. We identified differentially expressed genes between classes using the non-parametric rank-product method (p-value <0.05 and 0.01) and log2 expression values for each gene were generated.
