Hybridisation
Washing
Scan cy5
channel
Scan cy3
channel
“Overlay
 images”
Quantify
pixel intensities.
Cell
population A
Cell
population B
RNA
extraction
A
A
B
B
Reverse
transcription
A
A
B
B
Klenow
label
incorporation
Sample B labelled
with cy3 dye
Sample A labelled
 with cy5 dye
Expression Microarray Experimental overview
As a quick overview of the experimental procedures,
Firstly take two samples, these could be two cell populations, tissues disease states etc
Extract the RNA, There are several methods depending on amount of starting material, however past work has shown that this is one of the largest sources of variation in the experiment. RNA quality is very important and reliable data is reliant on good quality RNA

Next the RNA is reverse transcribed to cDNA, depending on the amount of RNA this may involve an amplification step.
The two samples are then labelled with one of two different flourophores cy5 and cy3 mixed then applied to the array and hybridised overnight

The slide is then washed and scanned, to produce two tif files, one for each of the samples and flourophore used.
The images are then overlaid and the pixel intensities for each probe quantified