1. Overview of Hybridization, Stringency, and Genechip Processing

2. The following hybridization mix is prepared for each sample
Fragmented cRNA ug ul
Control B2 Oligo 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] ul
DMSO 10 ul
2x Hybridization Buffer ul
Water ul
Denature 99C
10 minutes
Inject into
GeneChip

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

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 prevents:
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. Factors Influencing Stringency
Intrinsic factors   GC rich nucleic acid more stable because of triple H-bond  
Degree of complementarity
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. Steps in the Staining Protocol
Rinse away unhybridized FcRNA target
Stain with Streptavidin PE [SAPE]
Grand Total MW
(Minimum)
292,800
150,244
735,844 Da
WOW!!!
Stain with Biotinylated IgG anti-SAPE antibody
Stain AGAIN with Streptavidin PE [SAPE]
Rinse throughly

11. The Staining Chemistry for Affymetrix Genechip

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

13. Fluorescent Spectrum of Phycoerythrin
Stoke shift
Emission
Excitation Wavelength

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

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

16. Castleton State College-GeneChip Image Data
csc 1 csc 2 csc4
csc csc 8

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

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

19. How well do the sample types correlate ?