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Our lab is making a library from yeast total RNA. We have tried NuGen kit, and got huge rRNA peaks in the samples. We then tried Illumina's RNA-Seq and got no cDNA at all. Then we tried Ambion's mRNA purist to pull out just the Poly A, and are still getting significant rRNA carryover. Has anyone else had such problems with yeast RNA samples?
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Hi, I have been having this kind of problem as well isolating human polyA mRNA using Qiagen oligotex. Have you tried doing two rounds of purification? One of the Qiagen boffins has suggested that the rRNA contamination after purification might have something to with rRNA associated with translation of your mRNA... Do you have some pics of your bioanalyzer traces that show you your contam?
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