I'm having problems with my RNA extraction with Trizol.
Basically, I'm growing my plants in semi-sterile pouches (basically a plastic bag with a filter paper that allows the shoot to go up and the root go into the filter supplemented with a saline solution) and inoculating my bacteria in the filter paper. One week after I cut the roots, shake them, centrifuge and store the pellet at -80°C. So, in my samples there is no soil involved. I also checked the colonization and I have up to 10^8 bacteria per gram of root (so, more than enough).
But, I think the exudates from the plant are somehow inhibiting the Trizol extraction and I don't know how to fix it. Any ideas? :D
Maybe the plant secretes secondary metabolite that usually interrupt with RNA extraction. maybe u want to look at CTAB-based method?
Do enrichement of the bacteria isolated from the roots of the plant. The extract RNA using the bacteria protocol.
María, are you using a kit?? which one is? do you have a control to know if it is really working? or is a conventional method? sometimes in these cases is better the conventional o mix both.
Hi Pili,
How long last the root shaking? I advice to cryogenize your sample befor to stock them at -80°C in order to stop all active compounds in your sample.
These questions may seem stupid:
- Are you sur that all of your solutions are RNase free? (Autoclaved twice etc...)
- Performing all step at low temperature (keep tube and solution in ice etc...)
Most of the secondary metabolite or exudat might be lost during phase separation. I think RNA are present, and you probably lost it during a step. A tips which work well when I performed this kind of extraction in my former lab is the phase lock gel tubes.
Hi, everyone, thanks for the answers.
-Farhan: thanks, I will have a look to that
-Ag Muhammad: what do you mean by bacterial enrichment? Can you explain a Little bit more :)?
-Zayda: No, I'm using the Trizol-Chlorophorm protocol and It worked in other samples. So, the protocol itself it's not the Problem.
-Jordan: Yeap, I shake the samples 20 min in cold and I freeze the pellet at -80°C and all the solutions/material are RNAse free and clean (or that's what the brand claim ;p). That's not the Problem because I'm having good results with other Kinds of samples so my guess is that the plant are producing something that inhibits the reaction. I will check the phase lock gel tubes (no idea what are they).
Thanks everyone for the tips.
if you have the time, just spike your samples with something like E. coli from a normal NA plate. The idea being that you can identify at which step you're losing the RNA. Also, if you are sure it is the plant that is interfering, you may want to try a kit as they can really clean some of the nasty stuff out.
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