Issues with Cy5 labeled RNA - (Jul/05/2012 )

I have been using the Mirus Direct labeling system to label my RNA with Cy5. I keep having issues with one of my samples. My advisor says that my pmol per array should be between 35-55 and that my micrograms per array should be between 1-2 in order to use my samples on combimatrix chips. The sample I am having trouble with has an FOI (frequency of incorporation) of 26.6. When I calculate the possible hybridization volumes of sample to use, my pmol and ug/array do not fall in the range I need. She said it was probably due to the FOI being so high. She suggested repeating the EtOh purification steps in the labeling protocol to see if it would reduce the FOI. I have tried that and am still having the same problems. Does anyone have any suggestions or thoughts? Has anyone tried using RNeasy columns with Cy5-labeled RNA?

Rneasy columns should be fine with Cy5. Minelute columns are commonly used and I use Zymo RNA C&C -5 columns routinely.

If you are incorporating too much Cy5, more purification isn't going to help (Cy5 is covalently bound to the RNA). I don't have experience with the Mirus kit; I'd suggest consulting the manual to reduce incorporation.

Cy5 is notoriously sensitive to ozone -- if your Cy5 signal is too low, ozone may be the problem.

Thank you! I really appreciate your help with this!