Expanding use of microarray systems for global gene expression profiling is leading biological research, providing diagnostic and prognostic value to physicians and facilitating target discovery during drug and vaccine development. There is strong interest in extending this microarray capability to more RNA-limited material to the point that a single cell can be profiled with confidence. The most widely used method for preparing samples for microarray analysis, which uses cDNA synthesis from mRNA followed by T7 RNA polymerase-based amplification, currently lacks the necessary sensitivity to meet these emerging needs. To overcome these methodological limitations, we propose to increase the final amplified RNA yield by 1) improving the efficiency of T7 RNA polymerase using structure-based engineering with directed evolution of function, 2) by optimizing the design of the promoter-template construct for specific amplification needs and 3) by re-standardization of cDNA synthesis for lower RNA inputs. The combined improvements are expected to lower the cell-limited RNA sample requirement by 100-fold compared to current, commercially available kits. This will enable microarray gene analysis with as little as 1 ng of total RNA with a single round of amplification or microarray profiling from a single cell (-10 pg of total RNA) after two rounds of amplification. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Technology Transfer (STTR) Grants - Phase II (R42)
Project #
2R42GM072412-02
Application #
7108369
Study Section
Special Emphasis Panel (ZRG1-GGG-J (10))
Program Officer
Portnoy, Matthew
Project Start
2004-08-01
Project End
2009-08-31
Budget Start
2007-09-29
Budget End
2008-08-31
Support Year
2
Fiscal Year
2007
Total Cost
$672,066
Indirect Cost
Name
Accacia International, Inc.
Department
Type
DUNS #
084994735
City
Austin
State
TX
Country
United States
Zip Code
78728