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. 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. Staining the biotinylated fcRNA
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]
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

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
Stoke shift
Emission
Excitation Wavelength

16. The Scanned Yeast Array 220,000 probes 6,400 genes 11 um features 25 bp Sense DNA Oligo’s

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

18. SMC-2007-GeneChip Image Data

19. 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

20. QC Report From Genechip
Actin Housekeeping Control 3’-5’ Ratio
TATA BP

21. How well do the sample types correlate ?