Expression analysis overview:

From labeling to hybridization  >>  Probeset overview

Click on thumbnails to enlarge

Target labeling overview (for expression analysis):

To quantitate transcript abundance, first, totRNA is extracted from the sample and retro-transcribed to cDNA. An IVT reaction, using cDNA as template, is then performed. To obtain biotin-labeled cRNA molecules, a fraction of biotinylated nucleotides is used in the IVT reaction. The labeled cRNA is called "target".

    
GeneChip array manifacturing:

Affymetrix technology uses a photolithographic/chemical coupled method to synthesize 25-mer oligonucleotides on a silica wafer.
Oligo sequences are used for expression, genotyping or resequencing studies. 		   
Expression analysis target labeling overview:

In every single cell present on the array surface, millions of copies of the same 25-mer oligonucleotide molecule are synthesized.
Half a million different cells are present on a GeneChip.

Sets of cells define a probeset.

    
Target-Array hybridization:

During the hybridization process, biotinylated cRNA molecules from the sample to be analyzed, are available for binding to the GeneChip surface.
They will bind to the oligonucleotide containing complementary sequences.		    
Quantitation of hybridized target:

cRNA molecules will bind to their complementary sequences on the GeneChip surface, proportionally to their abundance in the hybridized sample.

The higher the number of bound cRNA molecules, the higher the signal intensity detected by the scanner after photochemical reaction.

    

Overview:

Biotinilated cRNA molecules are synthesized from the starting sample and allowed to hybridize to their complementary sequences on the GeneChip surface.
Through a photochemical reaction, the target bound to the array surface emits light when excited by the scanner at appropriate wavelenght .
The emission intensity collected by the scanner is proportional to the amount of target bound to the specific coligonucleotide.
Calculations are made using GCOS algorithm.