We are planning to do mutational analysis using Droplet Digital PCR and Cast PCR, can anybody suggest the best extraction methods for low ctcs (5-2000) cells?
Have you considered using commercial kits? There are some at the market.. and it is quite cheap...
You may perform general extraction of RNA/DNA from plasma. There is one critical moment in order to escape false positives: to remove CD34/CD35 stem cells, that share similar features with tumor cells. One approach (expensive) - to use sorter. 2-nd: to use commercial magnetic particles with antibodies.
Thank You for the reply. We have been using Nucleospin and Picopure for DNA extraction and Qiagen kit for RNA. I wanted to know if there is any better kit available to reduce the loss.
as u know CTCs are really rare so at the first u have to isolate them with good approaches like FACS or MACS technique
after that you can extract DNA or RNA with the better efficacy...
what the isolation method did u chose?
Hi Fatemeh, We have developed an instrument called Apostream to isolate CTCs. We have been getting good recovery. It is just the downstream process we are skeptical about.
Maxim,
Thank You for the reply. Will look into the kit that you mentioned.
if u had good recovery as Maxim said you can use kits for low dna/rna isolation...
good luck :)
this paper maybe helpful http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310221/
To Fatemeh
Will you please help me to know the mechanish of FACS for iso;ation of CTCs.
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