The long-range goal of this project is to develop new fixation methods for cell preservation that are compatible with molecular analyses used to study cellular proteins, DNA, and RNA. The present application seeks to characterize the structures that are formed between cellular components following treatment with aqueous buffered formaldehyde (neutral buffered formalin), the reagent that is currently the most common fixative agent. It is our contention that a full characterization of these structures will aid in the design of new fixatives and may allow us to develop methods to reverse the formalin linkages. To accomplish this goal we will first use purified systems that model intracellular DNA and RNA to determine the number and types of linkages formed by neutral buffered formalin treatment between proteins and nucleic acids. Once these analytical methods are worked out in these simple systems, they will be extended for formalin-fixed cells. The formalin-fixed model systems will be also be used to provide templates for PCR and RT-PCR analyses to allow a study of how this treatment inhibits these processes. As a part of these studies, attempts will be made to develop methods that might be used to reverse the formaldehyde adducts that have formed to the nucleic acids to generate better templates from fixed cells and a study to determine if the formaldehyde adducts induce instability in RNA. Once the chemistry of formalin fixation and the mechanism of PCR and RT-PCR inhibition are understood, future studies, not part of this application, will use this information to develop new fixative agents that do not interfere with molecular analyses or which can be chemically treated to reverse or cleave the fixation linkages.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21CA091180-01A1
Application #
6439029
Study Section
Special Emphasis Panel (ZCA1-SRRB-Y (O2))
Program Officer
Bledsoe, Marianna
Project Start
2002-04-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
1
Fiscal Year
2002
Total Cost
$146,917
Indirect Cost
Name
Wayne State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202