Extraction of RNA and reverse transcription:
The key reagent in transcriptional profiling is the RNA harvested from an organism. This RNA
is used as a template for reverse transcription to make cDNA for competitive hybridization against
the affixed probes on the microarray. RNA is best extracted from flash-frozen pellets of tissue
or culture grown in meticulously maintained common garden conditions. The flash-frozen matter
should have its RNA extracted in a manner that will not result in mRNA degradation. A detailed procedure
of mRNA extraction can be seen and downloaded here.
Cyanine Dye Coupling:
NHS dye may be bound to cDNA via amino-allyl-dUTP residues by raising the pH. To
10–13 µl of purified concentrate, 0.8 µl of 1 M NaHCO3 pH 9
can be added, with an appropriate NHS-cyanine dye aliquot. This coupling reaction is incubated
in the dark at 25° for 75 min and then stored in the dark at 4° and used in less than 24 h.
The labeled cDNA may then be purified with a QIAquick column. This elution of about 55 µl of
purified cyanine-labeled cDNA may also be stored at 4° and should be used in less than 24 h.
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