Hi everyone,
I’m isolating RNA for mRNA sequencing, and I need to perform a DNase I digest to remove genomic DNA before sequencing. I’m wondering if I can do the DNase I digest after RNA purification in solution using the Thermo kit (Cat No. 18068015), or if I’m required to do the digest on-column during RNA isolation.
I’m concerned that the buffer used for the in-solution digest might affect the RNA integrity or the sequencing results. Has anyone had experience with this? What would you recommend?
Thanks in advance!
You should not submit samples for sequencing that contain DNase, if that's your question. However it's very common to treat RNA with DNase in solution, and it has no effect on RNA integrity. Simply clean up the RNA afterwards to remove the DNase and resuspend in whatever buffer you like (eg column clean up or ethanol precipitation or beads).
Dear Ciaran,
thanks a lot for your answer. If a cleanup step is required we will just go with on column digest.
Yes, you can digest your RNA samples with DNase 1 in solution before performing mRNA sequencing. DNase I digestion is commonly accepted as an efficient and thorough method to eliminate genomic DNA (gDNA) contamination from RNA samples, which is crucial for mRNA sequencing to avoid interference from gDNA. This method is more efficient than column-based methods and ensures that the RNA sample is free from DNA contamination, which is essential for accurate and reliable mRNA sequencing results. Genes expressed at low levels raise false discovery rates in RNA samples contaminated with genomic DNA
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