How to extract DNA from fixed pathologic specimens for MSP - (Jan/05/2006 )
We have some old samples which are fixed pathologic specimens. Now we found the gene we are interested in can be methylated. So, we wonder if the gene is methylated in the samples. But unforunately, all we have is the fixed pathologic specimens.
My question is , how can we extract the DNA out of the fixed pathologic specimens? (we plan to extract the DNA, then modify, then do MSP.)
We are unable to locate any lab here can do in-situ MSP, so that can't be done. (We also thought of using laser to extract the specimen (LCM) but can't find any rescource either.)
Any suggestions? Thanks!
Hi Mlab,
That should not be a problem.
The first thing is to get DNA from the specimens. Are your specimens in paraffin blocks or in slides? I have extracted DNA from paraffin blocks as well as from a few cells dissected from slides. Here are some protocols you can follow to extract DNA. Since bisulfite modification doesn't require much DNA,
Regarding in situ MSP, I guess few people has ever used it since it was first reported about 5 years ago. Is it is too hard to do or unproducible? who knows.
If you have access to laser microdissect equipment that is great. If not, you can do microdissection under a microscope using a scaple. You can use a HE stained slide as a template and dissect on unstained slides, or dissect directly on stained slides. It takes some time to coordinate eyes and hands.
Good luck.
Sorry to dig out this post.
I'm intending to do something similar, except that I intend to do a BSP for the LCM samples.
my product size is about 450bp.
The BSP has been done succefully in cell lines already.
For LCM DNA,
I was able to get quite a lot of DNA.
Input DNA for bisulphite treatment: tried 1ug and 2ug
Have started with 2 samples and unfortunately failed.
I've read papers from groups who normally do shorter fragments (<200bp generally)
probably due to the fact that the extracted DNA tends to be in shorter fragments (forgot where I read this..)
Has anyone been successful with this?
I'm intending to do something similar, except that I intend to do a BSP for the LCM samples.
my product size is about 450bp.
The BSP has been done succefully in cell lines already.
For LCM DNA,
I was able to get quite a lot of DNA.
Input DNA for bisulphite treatment: tried 1ug and 2ug
Have started with 2 samples and unfortunately failed.
I've read papers from groups who normally do shorter fragments (<200bp generally)
probably due to the fact that the extracted DNA tends to be in shorter fragments (forgot where I read this..)
Has anyone been successful with this?
yes the DNA you are recovering from paraffin embedded samples is going to be quite fragmented already, and after the bisulfite the fragments will be even smaller.
Our lab uses smaller amplicons (as you said ~200bp) and sometimes it is also necessary to perform semi-nested PCR to get enough of the correct product to sequence.
the problem with using a large amplicon on fragmented samples is that you will stochastically amplify from a few pieces of DNA that are long enough (as opposed to amplifying a large pool of molecules) so your results may be heavily biased towards a single or few molecules
you can also try bisulfite treating for less time (=less fragmentation), however you need to make sure your primer design is quite specific for fully converted DNA, and check your sequences to make sure all non-CpG cytosines have been converted!
good luck!
My question is , how can we extract the DNA out of the fixed pathologic specimens? (we plan to extract the DNA, then modify, then do MSP.)
We are unable to locate any lab here can do in-situ MSP, so that can't be done. (We also thought of using laser to extract the specimen (LCM) but can't find any rescource either.)
Any suggestions? Thanks!
Check these protocols for some hints:
http://search.vadlo.com/b/q?sn=158621799&a...affin&rel=0
..
thanks cellcounter foryour valuable advice.
It seems like MSP may be a more practical approach in my case, as we are dealing with quite a number of slides.
THere's a kit from Zymo, EZ DNA Methylation-Direct™ Kit, which has increased sensitivity for FFPE samples.
Try to get the dealer in my country to give me a free sample for trial 