Microarrays
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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 labelled cDNA
-Yellow: gene expressed in both reference and test samples. - Green: gene expressed only in reference sample (i.e. down-regulated in the test sample). - Red: gene expressed only in test sample (i.e. up-regulated in the test sample).
Red Cy-dye labelled cDNA
Co-Hybridisation with microarray Laser scanning [5-10um resolution]
-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 labelled cDNA
-Yellow: gene expressed in both reference and test samples. - Green: gene expressed only in reference sample (i.e. down-regulated in the test sample). - Red: gene expressed only in test sample (i.e. up-regulated in the test sample).
Red Cy-dye labelled cDNA
Co-Hybridisation with microarray Laser scanning [5-10um resolution]
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