Hi everyone,
We have isolated total RNA from our samples using Qiagen RNeasy Plus Micro Kit (which includes genomic DNA removal columns), obtaining RNA yields of around 100 ng. These samples are going to be used for RNA sequencing, with the NEBNext Ultra II Directional kit and NEBNext Poly(A) mRNA Magnetic Isolation Module. Has anyone treated the RNA samples with DNase before proceeding to the library preparation? As we are worried with the risk of losing some of the RNA yield that we have, we would appreciate any recommendation about which would be the best way to collect back the RNA after the DNase treatment (magnetic beads or clean-up kits?), so if someone could provide us with a protocol for this step, it would be very helpful.
Dear Elisa,
I have done in vitro transcription for preparation of guide RNA and mRNA and for that after DNase treatment i did lithium chloride precipitation, it will work fine with RNA yields of 100ng but may not efficiently precipitate RNA smaller 300 nucleotides. Or else you can use some kit but i don't have any idea about the magnetic beads. Hope it helps
Hi, I don't know whether what I did is totally the same as yours. We need to synthesize double strand cDNA for sequencing, which we call it as transcriptome. We use TRIzol(kit) to extract the total RNA, then do the on column digestion with DNase as the protocol suggests. Later, we would run PCR to confirm that there is no DNA contamination.
In your case, you can use on-column DNase digestion with Qiagen's kit, but your total RNA quantity is really less, i don't know how much you can recycle after the DNase treatment.
Thanks to both.
Indeed Qihui, small amounts of RNA are our limitation.
Best,
Elisa
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