Microarrays

-DNA microarrays are modification of the ‘Southern blotting’

technique which depends on blotting DNA on a membrane and
then detect hybridisation with probes (labelled oligonucleotides).

- Described for the first time in 1987, which used spotting of DNA
on filter papers. The small [Micro-] form of the technique was
developed in 1995, and the whole eukaryotic genome of
Saccharomyces cerevisae on a microarray was the first to be
published in 1997.

- DNA microarrays consist of small glass or silicon chips.

- A microarray contains a large number [thousands] of

oligonucleotides [genes], or cDNA spotted in arrays on the chip.

- The oligonucleotide spots affixed to the chip are called ‘Probes’,

each ~ 100-150um in diameter, while the labelled cDNA
(synthesised by reverse transcriptase action on the RNA isolated
from reference and test samples) is called the ‘Target’
Manufacturing
The probes are fixed to the chips by:
1- Mechanically spotted using an array of fine tips controlled by
robotic arm which can transfer the ‘pre-synthesized’ probes
from wells and spot them on a chip, the probes attach by
covalent bonds to the chemical matrix of the chip on the
designated place for each.
2- In situ synthesized arrays
A- Mask-based Photolithography (25 mer oligonucleotide)
which depends on using light to transfer the probes from a
photo-mask to a photo-sensitive chip such as Affymetrix Inc.
GeneChips [single sample labelling].

B- Mask-less Photolithography (24-70 mer) such as

NimbleGen Systems Inc. [dual sample labelling].

C- Ink jet in situ synthesis (20-60 mer) such as Agilent

Technologies Inc. or Oxford Gene Technology [single or dual
sample labelling].
 Steps of production
Reference sample Test sample ‘tumour biopsy’

RNA isolation

RNA
cDNA (or cRNA) synthesis by using reverse
transcriptase or RNA polymerase enzyme and
Cy-dye labelled dNTPs

Green Cy-dye Red Cy-dye

labelled cDNA labelled cDNA

Co-Hybridisation with microarray

-Yellow: gene expressed in both reference and
test samples.
Laser scanning [5-10um
- Green: gene expressed only in reference
resolution]
sample (i.e. down-regulated in the test sample).
- Red: gene expressed only in test sample (i.e.
up-regulated in the test sample).