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 ?