I'm performing simultaneous extraction of DNA / RNA / PROT with Trizol, in samples embedded in OCT. The results for RNA and PROT are good, but I'm having problems with the yield and quality of the DNA.
Does anyone know how to improve it? I try to reduce the amount of OCT in the cuts, but it still does not work. Is there any method to remove the OCT?
There is a discussion of a topic similar to this here:
https://www.researchgate.net/post/DNA_extraction_from_TRIZOL_sample.
Hi!
I read as "OCT hasn't harmful effect on tissue DNA extraction", so do not worry for reducing or some thing.
When you are able to get good amount of RNA & Protein, you should get equally the amount of DNA too. What I feel is hope you are collecting correct phase (interphase, Phase separation is crucial) carefully and following steps carefully for DNA isolation. Check once.
for removal of OCT ckeck the following link
http://waldman.ucsf.edu/Protocols/dna.frozen.htm
Good Luck!!!
I regularly extract DNA form skin tissue embeded in OCT without any problem, but I do not extract RNA and protien from the same sample. I am not worried about RNA or protein. Therefore, I work on room temperature. I leave frozen tissue in a sterile dish at room temperature for few minutes, add the ultrapure water to melt the OCT faster.Then I hold the tissue with sterile forceps and wash with more water then follow the phenol /chloform protocol for DNA extraction. If the sections are cut and mounted on slides then I use sterile scalpel to remove the tissue sections, collect the scraped tissue in a sterile tube, add initial DNA extarction buffer and centriuge it, tisuue sits in the bottom and OCT is dissolved in buffer which is removed by using micropipetes and discarded. Then fresh buffer is added following the phenol/chloroform DNA extraction protocol.
Good Luck!
Thanks to all three for your answers. I'm testing with some modifications (i.e. using a co-precipitant, reducing as much as possible the amount of OCT in cuts, etc) and taking into account your comments too (correct interphase separation, etc). If I get better yields I tell you.
Hi Victor,
You say your results for RNA are good - I'm curious as to whether you've had any issues extracting RNA from OCT-embedded tissue? I'm getting low 260/230 ratio (which could be phenol/guanidinium contamination) and a very strong peak in the 5s region on Bioanalyzer using RNA pico chips. Have you seen anything like this?
Hi Aedan,
With the RNA I have not had the problems you mention. I use the Tri reagent Ambion protocol with the modifications suggested for small samples. The quality is good, theQPCR validation experiments goes well and also expression arrays.
By the way, some of my samples were obtained in your hospital's Tumor Bank.
Hi Victor,
That's quite a coincidence! I actually work in the Tumour Bank, so it's good to know that we should definitely be able to get good RNA from our samples. I'm using Qiagen's miRNeasy kit, so I expect small RNAs to be present, but they seem to be more abundant than the 18s and 28s RNAs which doesn't seem right.
It seems that the choice of Bioanalyzer chip was the issue - I was using Pico chips, but when run on Nano chips the trace looks exactly like I would expect for total RNA.
@Victor, I am having similar issue. Trying to coextract DNA and RNA from same human fresh frozen tissues. RNA seems fine, but yields of DNA are very low. Could give some insights towards improving DNA yields using Trizol method? Thanks
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