Double stranded cDNA synthesis for use with Nimblegen microarrays (Kathryn)

Many people in the lab in the past have used custom built Nimblegen microarrays to assess gene expression. For this purpose, high quality double stranded cDNA libraries must be produced from your RNA extractions.

This cDNA synthesis protocol is somewhat more elaborate than others. If you need to synthesize cDNA for some purpose other than use with Nimblegen microarrays, I suggest considering other methods. The reagents here are quite expensive. You may wish to run half reactions, depending on how much final cDNA you need (in my case, > 0.5 ug) especially if your success rate is low (which is what I mainly did). The protocol for half reactions is available here: cDNA synth_half rxn

I have used this protcol with RNA produced using the basic RNA extraction method (see this post).

This protocol is adapted from the Nimblegen guidelines (Chapter 2, Steps 3 – 6), available here: Nimblegen Users Guide

One modification of the Nimblegen protocol is the addition of Superase (Ambion) prior to first strand synthesis, in an effort to prevent RNA degradation during heating.