I am trying to extract RNA from preserved samples of Serratia marcescens. The cells are preserved in low concentrations, and cannot be cultured due to experimental constraints, therefore I am trying to optimize an extraction protocol for low concentrations.
I have tried using the Qiagen RNeasy Micro Kit, but cannot get the cells to pellet by centrifugation, therefore the RNA yield is very low (>1ng). Because I cannot get an initial pellet, I opted to use the fluid sample protocol in the MasterPure complete kit from Illumina, and get a higher yield of RNA (20-30ng/microliter), but am still having issues with low 260/280 values. Adjusting the DNase digest has helped to increase the 260/280, but it's still quite low around 1.65.
Does anyone have any suggestions for how to extract RNA from this small sample? Or suggestions on how to get a cell pellet from these cells?
Thanks for the reply. We have tried spinning at different speeds at 4C, including 5000xg up through 15000xg for 5-10 minutes and still cannot get a pellet. The bacteria is suspended in 1XPBS, and separately we have stocks suspended in glycerol that will need to be analyzed as well.
I am going to try using a membrane filter tomorrow, but am worried about loss of cells with this method.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA