Hi!
I have samples with callus remainings and bacteria that have been cultured on a callus solution. Now I want to isolate only the RNA from the bacteria to eventually only get mRNA from the bacteria to be able to RNA-seq. Now the problem is that the remainig callus appears to contaminate/overrule the RNA-seq data for now. So therefore, we would like to isolate the bacterial mRNA and remove all the mammalian parts.
If anyone knows something, let me know! :)
Thanks in advance!
Mammalian cells tend to be bigger than bacteria. You could try a trypsin digest to form single cells which you can then select using an appropriate sized filter mesh. This will not remove all contaminating mammalian RNA but it should at least enrich for your bacteria.
Not 100% sure but you can look into cell Separation to physically separate bacterial cells from the callus remnants using techniques like centrifugation followed by filtration (suitable for filtering out larger callus remnants). This will improve the over all enrichment of bacterial RNA.
You can possibly try a bacterial lysis buffer to break open the bacterial cells and release the RNA. Make sure the lysis buffer is optimized for bacterial cell disruption.
Also, look into ribosomal RNA (rRNA) removal kits like Ribo-Zero or RiboErase. Using Ribo-Zero or RiboErase kits with probes specific to mammalian rRNA, you can deplete the mammalian rRNA fraction, leaving behind the bacterial RNA and any other non-rRNA species. This approach enables you to focus on bacterial gene expression while minimizing the interference from mammalian RNA.
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