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 ?