Should we perform a denaturation step (65C degree treatment)?
3. My recollection from our discussion is that you require 5ug of sample RNA. Is this correct? What volume should it be in? (The final elution step of the kit I’m using leaves the sample in 70-80 microliters of buffer – should we EtOH precipitate this and bring it up in a lesser volume?) A: 1. No DNase is necessary unless you think there is significant contamination, enough to affect nucleic acid quantitation. First strand synthesis is from a polyT primer using reverse transcriptase, and amplification is by in vitro transcription, so there is little chance of genomic DNA contributing to the product. 2. No need to denature, this is done during primer hybridization. 3. The minimum for a standard Affy sample is 5ug at 0.5ug/ul. You may want to add Ambion Superase-In or similar to the eluate. If you elute in a buffer, EtOH precipitate with RNase free glycogen or PelletPaint, wash, and resuspend in water. Don’t let the pellet dry out completely. You could also use a Microcon3 or similar con
