strain: FVB/NJ genotype/variation: MMTV/c-MYC transgenic phenotype: tumor-bearing transgenic mouse tissue: total white blood cells (WBCs) data set: training
Extracted molecule
total RNA
Extraction protocol
Samples were collected using the submandibular (cheekpouch) method. Briefly, the vein that drains the face and cheek area was punctured by a lancet (GoldenRod animal lancet, MEDIpoint, Inc., Mineola, NY) and blood was captured in BD Microtainer™ tubes with potassium-EDTA anticoagulant (Becton Dickinson, Franklin Lakes, NJ). Tubes were immediately inverted 10-12 times and placed on ice. Following erythrocyte lysis, total RNA was isolated from the leukocyte pellet using the QIAamp RNA Blood Mini Kit (Qiagen, Valencia, CA). Alternatively, the leukocyte pellet was immediately lysed, homogenized and stored at -80°C. RNA was isolated at a later date using the protocol described above or the adapted protocol associated with the QIAamp RNA Blood Mini Kit for use in the QIAcube (Qiagen, Valencia, CA).
Label
biotin
Label protocol
Purified total RNA (200 ng) was amplified using NuGEN Ovation™ RNA Amplification System (NuGEN Technologies, Inc., San Carlos, CA). Biotinylated cRNA were prepared according to the standard Affymetrix IVT 1-cycle protocol from 2.5 ug total RNA.
Hybridization protocol
Following fragmentation, 15ug of labeled cRNA were hybridized on Mouse Genome M430 2.0 GeneChips for 16 hours at 45°C. GeneChips were washed and stained using the Biomex FX liquid handler.
Scan protocol
GeneChips were scanned using the Axon Genepix Scanner (Genepix 4000B).
