I've performed an assay in which I've injected 106 cells into the tail vein of nude mice and I'm trying to detect which cell model I injected had a greater invasive potential. I've already tried human Alu qPCR and the background seems to be too strong to overcome. I've also performed H&E staining of lung sections and can visually see differences in lung architecture in the different cell models. I'm just really in need of a more quantitative approach...any and all suggestions would be incredibly helpful! Thanks! ~ Andrew
Please review the following for references
An In Vivo Mouse Model for Human Prostate Cancer Metastasis
Neoplasia (2008) 10, 371–379
Abstract
We developed a sensitive real-time polymerase chain reaction (QPCR) assay that allows us to track early lodging/homing events in vivo. We used this technology to develop a metastasis assay of human prostate cancer (PCa) growth in severe combined immunodeficient mice. For this purpose, marked human PCa cell lines were implanted subcutaneously or in the prostate (orthotopically) of severe combined immunodeficient mice as models of primary tumors. Mice were then sacrificed at various time points, and distant tissues were investigated for the presence of metastatic cells. At 3 weeks, a number of tissues were recovered and evaluated by QPCR for the presence of metastatic cells. The data demonstrate that several PCa cell lines are able to spread from the primary lesion and take up residence in distant sites. If the primary tumors were resected at 3 weeks, in several cases, metastastic lesions were identified over the course of 9 months. We propose that this new model may be particularly useful in exploring the molecular events in early metastasis, identifying the metastatic niche, and studying issues pertaining to dormancy.
nested pcr has very great sensitivity...find any target dna which differs between human and mouse and amplify with human specific primers. dilute the product1;1000 and reampllfy with 10-20 cycles with the internal primers which,ideally, should also be human specific although it is less important as you already have a highly enriched target dna
I wouldn't give up on the Alu PCR. It should be among the most sensitive approaches. you clearly need to use primers that do not amplify mouse B1 repeats or 7SL RNA gene. Have you tried primer sets set up by Sinha (PMID:
12672420), These focus on some of the youngest and most distinct subfamilies of Alu elements. Adding droplet digital PCR as the analytical technique would not only make the quantitation more robust, but also decrease background.
I'm currently working with our DNA core facility here to optimize the Alu PCR we have in house. I've purchased a new set of primers and dual quenching probe to aid in specificity. Thanks to all for great suggestions. I'll certainly look up the Sinha primer sets (thanks Prescott for the ref) and also give a look into nested PCR (thanks, Paul). ~ Andrew
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