Year of Award:
Molecular & Cellular Analysis Technologies
Other PI or Project Leader:
UNIV OF MARYLAND, COLLEGE PARK
The aim of the proposed research is to demonstrate a technology to visualize the spatial distribution of genes and gene methylation in tissue sections. Our novel approach combines the key advantages of existing techniques: the amplification of in-situ PCR, the per-few-cells resolution of laser capture microdissection, and the multiplexing of FISH. We have already demonstrated that our technology can extract, amplify, and detect DNA in a spatially resolved manner using a substrate with mini-vials (so to a resolution of 1.6 millimeter per spot). Our aims now are to Aim 1 (achieve DNA methylation): Map GSTP1 promoter methylation over whole tissue sections from prostates with cancer. Aim 2 (achieve high spatial resolution): Miniaturize the technology to achieve a resolution of 100 ¨m. Aim 3 (multiplexing): Map multiple genetic alterations at once. We will demonstrate 3 but hundreds at once are possible in principle. It is also possible to spatially map gene expression (mRNA) by reverse-transcriptase PCR, and to quantify the amount of mRNA (by quantitative real-time PCR). Though we have initial results for both these directions and the path to them is open, we must meet aims 1 to 3 before we can move on to these more challenging areas. Consequently, multiplexed mRNA mapping and quantification will be done in future work. We will demonstrate a technology to spatially map genetic changes in tissue sections. Our method will allow the mapping of gene deletions, mutations, insertions, and methylations (silencing) in tumors, neighboring abnormal cells, and surrounding healthy tissue. The technology will be validated on frozen and paraffin fixed tissues supplied to us by Dr. Emmert- Buck at NCI and Dr. Olga Ioffe at UMB Medical School.