Hello,
we sequence long non-coding RNAs and as the first step of the whole process we perform rRNA depletion. To this date, we have used two rRNA depletion kits including GeneRead rRNA Depletion Kit (Qiagen) and RiboCop rRNA Depletion (Lexogen). Although the % of rRNA after depletion should be in both cases up to 3%, we observed this between 10% and 50%. Total RNA is isolated using mirVana miRNA isolation kit (Ambion) from the tissues stored 24h in RNAlater (4°C) before deep-freezing (-80°C).
Do you have similar experience? Can anyone advise me on how to reduce the percentage of rRNA at least below 10%?
My colleagues have had success with using a double depletion method. I assume they just deplete twice, using the normal method, though I don't know the specifics. Might be something to look into?
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