Simultaneous Fluorescent Detection of Two mRNA Targets by High Performance Signal Amplification

Abstract

Development of drug candidates, which modulate cytokine responses or metabolic pathways by targeting gene expression, requires analytical systems that measure specific messenger RNAs rapidly and accurately and that can be adapted to high throughput screening operations. Chromagen’s High Performance Signal Amplification (HPSA), system, a DNA probe hybridization method, has been enhanced to measure two different mRNA targets simultaneously in the same micro-plate well. Multi-target testing using HPSA takes advantage of a family of low molecular weight fluorescent dyes with large quantum yields, which exhibit distinct excitation and emission wavelengths. A two-target model system using cultured human THP-1 cells examined IL-1b mRNA induction in response to bacterial lipopolysaccharide (LPS) exposure and also monitored intrinsic b-actin mRNA as a housekeeping gene transcript. An oligonucleotide DNA probe complementary to IL-1b RNA was labeled with a reporter ligand while a b-actin DNA probe was linked directly to an enzyme. Sample mRNA was captured onto the surface of micro-plate wells and hybridized to both DNA probes. After removing nonhybridized probe, a second enzyme conjugate that specifically recognizes the IL-1b DNA probe reporter ligand was allowed to bind. The two enzyme systems were distinguished by using substrates labeled with different fluorescent tags (one for IL-1b and the other for b-actin). Resolution of the two fluorescent products was carried out with Chromagen’s high-sensitivity, photoncounting fluorometer. This system was capable of detecting synthetic RNA targets for IL-1b and b-actin in the attomole range. Authentic b-actin mRNA was measured in 50,000 uninduced cells and this signal could be specifically competed away by addition of excess unlabeled b-actin probe during hybridization. A time course of IL-1b mRNA induction in THP-1 cells by LPS revealed that peak induction occurred after 2-3 hours. The bactin mRNA level showed an initial decrease, but remained relatively constant throughout the remaining time points. Results obtained with the dual detection format paralleled those generated when each target was measured separately in its single-target gene expression assay. Chromagen’s HPSA two-target system not only augments screening information, but also saves testing time and conserves reagents. The potential of this system is being explored with different gene targets of therapeutic value and other housekeeping genes. This presentation was given at the 1999 International Symposium for Laboratory Automation and Robotics held in Boston, MA, October 17-20, 1999. The full manuscript is available on CD-Rom and can be acquired by contacting Christine O’Neil, 508-497-2224; email islar@islar.com or access the ISLAR website at www.islar.com.