72 hours after transduction with Mn1, gfp positive bone marrow cells were sorted and RNA isolated
Extracted molecule
total RNA
Extraction protocol
Quiagen kit
Label
biotin
Label protocol
cDNA target was synthesized, fragmented, biotin-labeled using WT target labeling and Control Reagents (Affymetrix, vat. num. 900652) starting from 200ng total RNA, according to the procedure described in the geneChip Whole Transcript Sense Target labeling Assay Manual, Version 4 (Affymetrix, santa Clara, USA). cDNA was fragmented and the resulting fragments of approximately 40-70 nucleoides were monitored with Bioanalyzer using the RNA nano 6000 Chip.
Hybridization protocol
The hybridisation coctail (80microliter) containing fragmented biotin-labeled target DNA at a final concentration of 25nanogram/microliter was transfered into Affymetrix GeneChip Mouse Gene 1.0 ST Arrays and incubated at 45 degrees on a rotator in a hybridisation oven 640 (Affymetrix) for 17 hours at 60rpm. The arrays were washed and stained on a Fluidics Station 450 by using Hybridisation Wash and Stain Kit (Affymetrix, cat. nu. 900720) using fluidics procedure FS450_0007.
Scan protocol
The GeneChips were processed with an Affymetrix GeneChip Scanner 3000 7G. DAT image files of the microarrays were generated using Affymetrix GeneChip Command Console (AGCC, version 0.0.0.676).
