Fisher rat BM cells were plated on 600w glucose DMEM, 40% MCDB-201, 1X insulin-transferrin-selenium (ITS; Sigma), 1X LA-BSA, 0.05x10-6M dexamethasone (Sigma), 10-4M ascorbic acid 3-phosphate, 100 units of penicillin, 1,000 units of streptomycin, 55 ?M 2-mercaptoethanol, 2 0.000000BS (HyClone; Lot ANL19977), 10ng/mL hPDGF-BB, 10ng/mL mEGF, and 1000 units/mL mLIF in a humidified, 502 and 6% CO2, 37oC incubator. After 4 weeks, CD45+ cells were depleted using a MACS separation CS column, and cells replated at 10 cells/well. Expansion was done by trypsinizing (0.05% trypsin) the cells and replating them every two days at a density of 200 cells/cm2. Cells at population doubling >100 were used for 3 RNA collections for gene arrays
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNAeasy microkit (Qiagen, Valencia, CA), DNAse treatment was performed on column
Label
Biotin
Label protocol
200 ng of total cellular RNA was subjected to double amplification. First round of IVT-based, linear amplification was performed using the RiboAmp OA RNA Amplification Kit (Arcturus, Mountain View, CA) followed by labeling with the Enzo Bioarray HighYield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) according to manufacturer's instructions
Hybridization protocol
Samples were hybridized to Affymetrix Rat Expression Set 230A chips (Affymetrix Inc), washed, and scanned at the University of Minnesota Affymetrix Microarray Core Facility as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Protocol EukGE-WS2v4_450
Scan protocol
Array was scanned at the University of Minnesota Affymetrix Microarray Core Facility as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Pixel Size 2.5, Filter 570, Scanner ID 50209060, Number of Scans 1, Scanner Type M10
Description
rat clone 2, biological replicate B, Affymetrix
Data processing
CEL files were loaded into GeneData Expressionist Refiner (GeneData, San Francisco, CA) to assess overall quality and obtain condensed single intensity values per probeset using the Microarray Analysis Suite Statistical algorithm (MAS 5.0). The mean of mean intensity values for all chips (192) was used to normalize the mean intensity of each chip. Normalization Target: 292, Tau: 0.015, Alpha1: 0.04, Alpha2: 0.06
