NSF Award
9560501
*** 9560501 Dhundale Messenger RNA (mRNA) analysis is a critically important technique in molecular biology. The goal of this project is the development of an ultra-sensitive method enabling direct measurement of endogenous mRNA in cell lines or primary cells present in a 96 well microplate. The procedure will negate the need for mRNA purification, thermocycling, or other forms of target amplification such as required with Q-PCR. A defined biotinylated DNA oligonucleotide probe will specifically hybridize to the target mRNA in fixed cells. Background or nonspecifically bound signal will be minimized by 1) requiring two specific hybridization events and 2) washing and subsequent release of the specifically bound probe which is then transferred and recaptured in a streptavidin coated plate. A sandwich-type assay will be employed with an ultra-sensitive, novel, enzymatic amplification system to increase the signal and yield a luminescent end-point. The method should be sensitive enough to measure an mRNA with ten copies per cell, 10,000 cells per well, i.e.- subattomole mRNA levels or 100,000 mRNA molecules per well. The method has generic utility since each new target mRNA simply requires synthesis of an appropriate oligonucleotide and use of standardized fixation, hybridization, and signal amplification procedures. Oncogene Science Inc. will offer reagent systems through the Research Products Division to provide a powerful tool for the molecular and cellular bioscience research community. ***
