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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA140068-04
Application #
8300976
Study Section
Special Emphasis Panel (ZCA1-SRLB-R (M1))
Program Officer
Sorg, Brian S
Project Start
2009-08-05
Project End
2014-01-31
Budget Start
2012-08-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2012
Total Cost
$128,040
Indirect Cost
$42,680
Name
University of Maryland College Park
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
Armani, Michael; Rodriguez-Canales, Jaime; Gillespie, John et al. (2009) 2D-PCR: a method of mapping DNA in tissue sections. Lab Chip 9:3526-34