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Overview of Hybridization, Stringency, and Genechip Processing. Denature 99C 10 minutes. Inject into GeneChip. The following hybridization mix is prepared for each sample. Fragmented cRNA 5ug 10 ul Control B2 Oligo1.7 ul - PowerPoint PPT Presentation
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Overview of Hybridization, Stringency, and Genechip Processing
The following hybridization mix is prepared for each sampleFragmented cRNA 5ug 10 ul Control B2 Oligo1.7 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 ulDMSO10 ul2x Hybridization Buffer 50 ulWater22.3 ul
RNA-DNA Hybridization
Probe sets: The DNA oligo probe is attached to the GeneChip via a silane bond
Targets:Antisense biotinylated cRNA
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
PNALNA
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 complementarityFactors Influencing StringencyExtrinsic 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
Stringency In Microarray Hybridization High stringency is obtained by: Low salt or buffer concentration High temperatureLow stringency is obtained by: Lowering the temperature of hybridization Increasing salt concentration [to a point]
High Stringency vs. Low Stringency
Processing the Yeast Genechip
Three Components to the Affymetrix GeneChip System
Hybridization oven -for hybridization of the target to the chipThe Fluidic Station- for staining
GS 3000 Scanner- for high resolution laser scanning of the stained chip
The Fluidics StationStaining 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
Steps in the Staining ProtocolRinse away unhybridized FcRNA targetStain with Streptavidin PE [SAPE]Stain with Biotinylated IgG anti-SAPE antibodyStain AGAIN with Streptavidin PE [SAPE]Rinse throughlyGrand Total MW(Minimum)
292,800150,244292,800735,844 DaWOW!!!
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 PhycoerythrinExcitation WavelengthEmission
The Scanned Array
500,000 probe features
24,000 genes
18 um features
25 bp Sense DNA Oligos
Microarray Images and QC -Good for seeing visual defects-Examining Borders, Chip ID, ControlsWhy do we look at this image?
Marlboro College-GeneChip Image Data
QC ReportCheck 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 RatioQC Report From Genechip
How well do the sample types correlate ?