Overview of Hybridization Stringency and Genechip Processing Genechip

Overview of Hybridization, Stringency, and Genechip Processing

Genechip hybridization preparation Fragmented c. RNA 2 ug Control B 2 Oligo 20 x Eukaryotic Control mix [bio B, bio C, bio D, Herring Sperm DNA [10 mg/ml] Acetyleted BSA [50 mg/ml] DMSO 2 x Hybridization Buffer Water 10 ul 1. 7 ul Cre] 1 ul 10 ul 50 ul 22. 3 ul Denature 99 C Inject into 10 minutes Gene. Chip

Genechip Hybridization -The Gene. Chip is placed in a hybridzation oven at 45 C -Hybridization occurs for 16 hour at 60 rpm -RNA-DNA Hybridization Targets: Antisense biotinylated c. RNA Probe sets: The DNA oligo probe is attached to the Gene. Chip via a silane bond

What is Hybridization Optimized Hybridization is the process of single stranded nucleic acids binding to another strand with identically complement sequence Types: PNA DNA to RNA to RNA LNA to DNA PNA to DNA LNA

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

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- Na. Cl, 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 temperature Low stringency is obtained by: Lowering the temperature of hybridization Increasing salt concentration [to a point]

Processing the Yeast Genechip

Steps in the Staining Protocol Rinse away unhybridized Fc. RNA target Stain with Streptavidin PE [SAPE] Stain with Biotinylated Ig. G anti-SAPE antibody Stain AGAIN with Streptavidin PE [SAPE] Grand Total MW (Minimum) 292, 800 150, 244 Rinse throughly 292, 800 735, 844 Da WOW!!!

The Staining Chemistry for Affymetrix Genechip

Scanning the Yeast 2. 0 Gene. Chip with the GS 3000 -Nd-YAG laser 532 nm -2. 5 u. M resolution

Fluorescent Spectrum of Phycoerythrin Stoke shift Emission Excitation Wavelength

The Scanned Array 500, 000 probe features 6, 600 genes 18 um features 25 bp Sense DNA Oligo’s

Microarray Images and QC Why do we look at this image? -Good for seeing visual defects -Examining Borders, Chip ID, Controls

JSC Microarray-Gene. Chip Image Data

QC Report Why do we look at the QC report? -Check 3’ to 5’ ratios of housekeeping genes-below 3 -Scaling factor-below 10 -Spike in control signal -Percent present-40 -60%

QC Report From Genechip Actin filament Transcription Initiating Factor IID

How well do the sample types correlate ?