Post on 15-Mar-2016
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Overview of Hybridization, Stringency, and Genechip Processing
The following hybridization mix is prepared for each sample
Fragmented cRNA 5ug 10 ul Control B2 Oligo 1.7 ul20x Eukaryotic Control mix [bio B, bio C, bio D, Cre] 5 ul Herring Sperm DNA [10mg/ml] 1 ul Acetyleted BSA [50mg/ml] 1 ulDMSO 10 ul2x Hybridization Buffer 50 ulWater 22.3 ul
Denature 99C
10 minutes
Inject into
GeneChip
Probe sets: The DNA oligo probe is attached to the GeneChip via a silane bond
Targets:Antisense biotinylated cRNA
RNA-DNA Hybridization
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 RNALNA to DNA PNA to DNA
PNA LNA
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 formationDisassociation of strands
Intrinsic factors
GC rich nucleic acid more stable because of triple H-bond
Degree of complementarity
Factors Influencing Stringency
Extrinsic factors
Experimentally introduced
TemperatureSalt concentration- NaCl, Na citrate, morpholinoethanesulfonic acidPresence of denaturing agents (e.g., formamide)Presence of high molecular weight polymers (e.g., dextran sulfate)Shear forcesMolecular tagging
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]
High Stringency vs. Low Stringency
Processing the Yeast Genechip
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!!!
The Staining Chemistry for Affymetrix Genechip
Scanning the Yeast 2.0 GeneChip with the GS3000
-Nd-YAG laser 532nm
-2.5 uM resolution
Fluorescent Spectrum of Phycoerythrin
Excitation Wavelength
Emission
Stoke shift
The Scanned Array
500,000 probe features
24,000 genes
18 um features
25 bp Sense DNA Oligo’s
Microarray Images and QC
-Good for seeing visual defects-Examining Borders, Chip ID, Controls
Why do we look at this image?
Castleton State College-GeneChip Image Data
csc 1 csc 2 csc4
csc 7 csc 8
QC Report
-Check 3’ to 5’ ratios of housekeeping genesWhy do we look at the QC report?
-Scaling factor-Spike in control signal-Percent present
GAPDH Control 3’-5’ Ratio
QC Report From Genechip
How well do the sample types correlate ?