Large-Scale Transcriptome Analysis
9
Day 1 afternoon
Thaw “control” or normotensive strain samples 1–4 and “experimental” or hypertensive strain samples 13–16. Apply the hybridization cocktails to microarrays and hybridize overnight
Day 2 morning
Prepare solutions for washing and staining. Process “control” or normotensive strain samples 1–4 and “experimental” or hypertensive strain samples 13–16 with the fluidics station. Scan these eight arrays
Day 2 afternoon
Thaw “control” or normotensive samples 5–8 and “experimental” or hypertensive strain samples 17–20. Apply the hybridization cocktails to microarrays and hybridize overnight
Day 3 morning
Prepare solutions for washing and staining. Process “control” or normotensive strain samples 5–8 and “experimental” or hypertensive strain samples 17–20 with the fluidics station. Scan these eight arrays
Day 3 afternoon
Thaw “control” or normotensive strain samples 9–12 and “experimental” or hypertensive strain samples 21–24. Apply the hybridization cocktails to microarrays and hybridize overnight
Day 4 morning
Prepare solutions for washing and staining. Process controls 9–12 and experimentals 21–24 with the fluidics station. Scan these eight arrays to complete the generation of experimental data
2.14 Materials
2.14.1 Hybridization Components, Stock Solutions and Buffers
Affymetrix manuals are available online and are very user-friendly. The protocols listed by Affymetrix should be strictly followed (http://www.affymetrix.com/support/technical/manuals.affx). All solutions should be prepared using ultrapure water (purified deionized water with a sensitivity of 18 MW at 25 °C), unless otherwise specified, and analytical grade reagents. Prepare all stock solutions at room temperature and store at the proper temperatures listed. 1. Water, Molecular Biology Grade (Fisher Scientific, Pittsburgh, PA). 2. Acetylated Bovine Serum Albumin (BSA) solution (50 mg/ mL) (catalog number 15561-020) (Life Technologies). 3. Herring Sperm DNA (catalog number D1811) (Promega Corporation). 4. GeneChip® Hybridization Control Kit (catalog number 900454) (Affymetrix, Santa Clara, CA) Contains the 20× Eukaryotic Hybridization Control Stock composed of premixed biotin-labeled bioB, bioC, bioD and cre, in staggered amounts, which is added directly in the preparation of the hybridization cocktail. These controls allow for monitoring of the hybridization process for troubleshooting. The kit also contains the Control Oligonucleotide B2 (3 nM) which is used for alignment of array probe cell features for image analysis.
