I am trying different methods for my enrichment culture which contain lots of Fe(II) and Fe(III) minerals (oxidized from 10mM FeCl2). The amount of total cell numbers are around 104-105 cells/ml, which is not a high cell number. We normally incubated them in 25 ml serum bottle under anoxic condition without any other organic carbon source.
I always have the positive control which is the same enrichment culture growing with acetate as organic carbon source. (105-106 cells/ml)
Previous technician tried "RNeasy PowerSoil Total RNA Kit" but does not work.
I did used other protocol accompany with the pre-treatment of iron-mineral removal method (with oxalate solution) to extract the RNA, and I did not get any RNA.
We also tried "TriFast" to extract the RNA, without washing out the Fe-minerals and with. We also tried 10 fold scale up (250ml of samples), the cell number should be approximately the same as positive ctrl. The positive control always work well. But the RNA conc. in my samples are always under the detection limit, which measure by Qubit Fluorometer. The weird thing is I got super high number of conc. from nanodrop quantification(but for the positive control super low conc.)
Would any of you please give me some guide or suggestion to modified the kit or suggest me other kits to extract the RNA from my enrichment culture (It was isolated from environment fresh water sediment. It contains several different organisms.)
We would like to further proceed meta-transcriptomic for this sample. It is very important to get nice quality of RNA.
Thank you in advanced for welling to help me to solve the problem and I am looking forward to discussions and answers!
A very detailed question, and it sort of intersects my fields of interest. But i don't have a clearcut perfect answer.
Problem with the setup could be that the columns you named are very VERY ionically sensitive. Therefor you use EDTA in the extraction buffer for the columns. I would say, either dilute your sample, (yes even more) before doing the extraction, and then doing PCR. Or you could use more EDTA. Although too much edta will push other elements out of solution which may make extraction of RNA less specific.
Other option would be using a non ionogenic extraction. Maybe one using antigenic binding, or using gels.
Another possibility is that the iron minerals interfere with the extraction process itself, and the RNA gets stuck within the bacterial cell. As some ligands or cell lysis proteins may be affected by it.
Hope this helps.
Hi, I am having exactly the same problem as yours! I am wondering if you have fixed the problem now?
Nanqing Zhou I tried several methods and also modified a bit. In the end I figure out one method works for my enrichment cultures. It's quite nice and stable method to get RNA from my samples. The reference of the method is in the attachment. I used up-scaled version. If you need modified version, feel free to contact me by Email. Good luck!
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