Quantitative Gene Expression Assays without Target Amplification

Abstract

Evaluating changes in gene expression is essential when identifying and validating new drug targets, however, the methods used to measure transcription are laborious, time-consuming and expensive ( e.g., RT-PCR, microarray or northern blot). High Performance Signal Amplification (HPSA™) gene expression assays quantitate particular mRNA targets directly in cell lysate samples using DNA probe hybridization and fluorescent signal amplification. The assay format eliminates the need for RNA purification prior to testing and does not require RT-PCR amplification. The HPSA™ protocol involves three steps carried out at 37°C in 96- or 384-well plates, making the technique amenable to automation. Cellular mRNA levels are quantitated relative to a standard curve consisting of purified in vitro RNA transcripts derived from cDNA clones. Assay sensitivity is in the low attomole range and can detect mRNA expressed at twenty copies per cell. A number of HPSA™ assays have been developed for cytokine, housekeeping and cytochrome P450 messages. Gene induction profiles were monitored in cell lines or peripheral blood mononuclear cells (PBMC) treated with activators such as phorbol 12-myristate-13-acetate (PMA), ionomycin or phytohemagglutinin (PHA). Recent validation studies demonstrated IL-9 mRNA induction in primary cultures of T-cells treated with PMA and anti-CD3. Similar testing with PBMCs showed significant IL-13 mRNA induction after 48 hours of treatment with PMA, thymosin and staphylococcal enterotoxin. Cytokine STATE® panels are being constructed according to functional categories such as innate or adaptive immunity to better characterize changes in transcription patterns during an immune response. The HPSA™ gene expression assays offer a rapid and convenient alternative to more cumbersome, expensive methods.