You recommend 25 ug of total RNA which is five times greater than recommended by Affymetrix. Why?
Labeled target for hybridization to spotted microarrays can be labeled using three different methods. 1) In direct incorporation a labeled nucleotide is incorporated during reverse transcription. 2) In indirect labeling an amino allyl modified nucleotide is incorporated during reverse transcription. After reverse transcription a fluorescent dye is chemically coupled to the amine group. 3) In dendrimer labeling an oligo d(T) with a 5′ sequence tag is used to prime reverse transcription. Following hybridization with the cDNA a second hybridization is performed with a fluorescently labeled dendrimer with a sequence complimentary to the tag on the primer. Each dendrimer can be labeled with many fluorescent molecules and so it can be very sensitive. We have found that dendrimer labeling (Genisphere) using about 1 ug of total RNA to be equally sensitive as direct labeling (Amersham) using 25 ug of RNA. The dendrimer labeling has the advantage of showing none of the dye bias observed with dir
