Overview%20of%20Hybridization,%20Stringency,%20and%20Genechip%20Processing

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Overview of Hybridization, Stringency, and
Genechip Processing
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Genechip hybridization preparation
Fragmented cRNA 2ug 10 ul
Control B2 Oligo 1.7 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 DMSO 10 ul 2x Hybridization
Buffer 50 ul Water 22.3 ul
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-The GeneChip is placed in a hybridzation oven at
45C-Hybridization occurs for 16 hour at 60
rpm-RNA-DNA Hybridization
Genechip Hybridization
Targets Antisense biotinylated cRNA
Probe sets The DNA oligo probe is attached to
the GeneChip via a silane bond
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What is HybridizationOptimized 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
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What influences hybridization? 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
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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
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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
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Processing the Yeast Genechip
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Steps in the Staining Protocol
Rinse away unhybridized FcRNA target
Stain with Streptavidin PE SAPE
Grand Total MW (Minimum) 292,800 150,244 292,800
735,844 Da WOW!!!
Stain with Biotinylated IgG anti-SAPE antibody
Stain AGAIN with Streptavidin PE SAPE
Rinse throughly
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The Staining Chemistry for Affymetrix Genechip
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Scanning the Yeast 2.0 GeneChip with the
GS3000-Nd-YAG laser 532nm-2.5 uM resolution
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Fluorescent Spectrum of Phycoerythrin
Emission
Excitation Wavelength
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The Scanned Array500,000 probe
features6,600 genes18 um features25 bp
Sense DNA Oligos
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Microarray Images and QC
Why do we look at this image?
-Good for seeing visual defects
-Examining Borders, Chip ID, Controls
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JSC Microarray-GeneChip Image Data
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QC Report
Why do we look at the QC report?

• Check 3 to 5 ratios of housekeeping genes-below
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-Scaling factor-below 10
-Spike in control signal -Percent present-40-60
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QC Report From Genechip
Actin filament
Transcription Initiating Factor IID
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How well do the sample types correlate ?