base.cbu.uib.no:PROT:Protocol-58 - base.cbu.uib.no:PROT:Protocol-58
The SuperScript? Indirect cDNA Labeling System is a highly efficient system for generating fluorescently labeled cDNA for use on microarrays in gene expression studies (De Risi et al., 1996; Eisen & Brown, 1999). It uses an aminoallyl-modified nucleotide and an aminohexyl-modified nucleotide together with other dNTPs in a cDNA synthesis reaction with SuperScript? III Reverse Transcriptase. After a purification step to remove unincorporated nucleotides, the amino-modified cDNA is coupled with a monoreactive, N-hydroxysuccinimide (NHS)-ester fluorescent dye. A final purification step removes any unreacted dye, and the fluorescently labeled cDNA is ready for hybridization to microarrays. This system uses 5?20 ?g of total RNA or 0.4?2 ?g of mRNA as starting material, and is compatible with Alexa Fluor? 555 and Alexa Fluor? 647 fluorescent dyes from Invitrogen, Cy3? and Cy5? dyes from Amersham Biosciences, or other monoreactive NHS-ester dyes from a variety of manufacturers.