I have a scarce amount of RNA (10 - 15 ng) and want to do both microarrays (Agilent 4x44k), where the recommended Cyanine 3/5-labeled linearly amplified aRNA is 825 ng. Also, I would like to perform qPCR on those samples.
I found that it is possible to preamplify RNA with amino allyl aRNA (AA-aRNA) and then use part of it for microarray aRNA labeling and part for cDNA synthesis for qPCR (Jeanty 2010). However, cDNA template from AA-aRNA performs worse than mRNA (higher Ct values) in qPCR.
Do you have experience with this or would you suggest something else?
HI,
I agree with Julie. I've used the same NuGen kit and it works great with minimal amounts of RNA.
It will give you cDNA that you can then use for either microarrays (I used Affy) and for 1PCR.
I found the 12 reaction kit but, if you plan things right and amplify more than a few at the same time, you can amplify up to 14-15 samples.
Good luck
Thank you all for your answers! The NuGen Pico kit will hopefully fulfil our needs :)
Since we used aRNA for hybridization up to now, is there any difference in binding of either aRNA or cDNA to oligo DNA arrays?
There's a product from a Company, Genisphere (genisphere.com) that I have used successfully in the past (called SenseAMP, and then RampUP, and I believe is now called Sensation Plus). Check it out. I hope this helps. All the best with your experiments. David
I never got around to use the WT-Ovation kit from NuGEN but did read about the good success possible with this kit. A paper with a very systematic analysis of its performance can be found on BMC Research Notes, Vermeulen et al 2009 : https://www.biomedcentral.com/1756-0500/2/235
Best,
~a.
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