1. Overview of Hybridization, Stringency, and Genechip Processing

2. The following hybridization mix is prepared for each sample Fragmented cRNA 5ug 10 ul Control B2 Oligo1.7 ul 20x Eukaryotic Control mix [bio B, bio C, bio D, Cre] 5 ul Herring Sperm DNA [10mg/ml] 1 ul Acetyleted BSA [50mg/ml] 1 ul DMSO10 ul 2x Hybridization Buffer 50 ul Water22.3 ul Denature 99C 10 minutes Inject into GeneChip

3. Probe sets: The DNA oligo probe is attached to the GeneChip via a silane bond Targets: Antisense biotinylated cRNA RNA-DNA Hybridization

4. Hybridization Optimized Hybridization is the process of single stranded nucleic acids binding to another strand with identically complement sequence Types:DNA to DNA DNA to RNA RNA to RNA LNA to DNA PNA to DNA PNA LNA

5. Stringency Stringency is a condition that causes a change in the local hybridization environment and “interferes” with the binding kinetics Stringency prevents:. Binding of non-complementary strands Self hybridization – hairpin formation Disassociation of strands

6. Intrinsic factors GC rich nucleic acid more stable because of triple H-bond Degree of complementarity Factors Influencing Stringency Extrinsic factors Experimentally introduced Temperature Salt concentration- NaCl, Na citrate, morpholinoethanesulfonic acid Presence of denaturing agents (e.g., formamide) Presence of high molecular weight polymers (e.g., dextran sulfate) Shear forces Molecular tagging

7. Stringency In Microarray Hybridization High stringency is obtained by: Low salt or buffer concentration High temperature Low stringency is obtained by: Lowering the temperature of hybridization Increasing salt concentration [to a point ]

8. High Stringency vs. Low Stringency

9. Processing the Yeast Genechip

10. Three Components to the Affymetrix GeneChip System Hybridization oven -for hybridization of the target to the chip The Fluidic Station- for staining GS 3000 Scanner- for high resolution laser scanning of the stained chip

11. The Fluidics Station Staining the biotinylated fcRNA An automated system to stain the target using streptavidin-phycoerythrin [SAPE], a biotinylated anti-SAPE antibody, and SAPE again… high and low stringency buffers are used

12. Steps in the Staining Protocol Rinse away unhybridized FcRNA target Stain with Streptavidin PE [SAPE] Stain with Biotinylated IgG anti-SAPE antibody Stain AGAIN with Streptavidin PE [SAPE] Rinse throughly Grand Total MW (Minimum) 292,800 150,244 292,800 735,844 Da WOW!!!

13. The Staining Chemistry for Affymetrix Genechip

14. Scanning the Yeast 2.0 GeneChip with the GS3000 -Nd-YAG laser 532nm -2.5 uM resolution

15. Fluorescent Spectrum of Phycoerythrin Excitation Wavelength Emission Stoke shift

16. The Scanned Array 500,000 probe features 24,000 genes 18 um features 25 bp Sense DNA Oligo’s

17. Microarray Images and QC -Good for seeing visual defects -Examining Borders, Chip ID, Controls Why do we look at this image?

18. Marlboro College-GeneChip Image Data

19. QC Report -Check 3’ to 5’ ratios of housekeeping genes Why do we look at the QC report? -Scaling factor -Spike in control signal -Percent present

20. GAPDH Control 3’-5’ Ratio QC Report From Genechip

21. How well do the sample types correlate ?