Direct Measurement of Multiple mRNAs in Nerve Growth Factor-Induced PC12 Cells Using Electrophoretic Tags to Monitor Biomarkers of Neuronal Differentiation in 96-Well Format

Pheochromocytoma-12 (PC12) cells recapitulate the program of neuronal differentiation by developing neurites after about 12 days of nerve growth factor (NGF) treatment. This model can be used to evaluate the neuroprotective/neurotrophic effect of compounds. Specific mRNAs such as cfos and c-jun are early biomarkers of the irreversible commitment into the differentiation program as they appear after only 30–40 min of NGF treatment. Monitoring the level of these mRNAs instead of the neurite outgrowth dramatically reduces the time needed to identify the drug potential of compounds. The electrophoretic tags, or eTag^™ reporters (ACLARA Biosciences, Inc., Mountain View, CA), are a new class of fluorescent reporters that have unique migration properties in capillary electrophoresis, which allows for their separation and identification. (The eTag Multiplex Invader^® Assay and products incorporate Invader^® technology and Cleavase^® enzyme licensed for use from Third Wave Technologies, Inc. [Madison, WI] for multiplexed gene expression applications.) Each eTag molecule used begins as a phosphoramidite that is incorporated into a specific oligonucleotide using standard oligonucleotide synthesis procedures. A set of distinct probes labeled with different eTag molecules can then be mixed together to simultaneously quantify the levels of different mRNAs from the same sample. When compared to existing methods for measuring multiplexed gene expression from the same sample, the eTag assay allows a direct quantification of the mRNA from cells without any extraction/purification and still provides multiplexing capability, high sensitivity, miniaturization, and reproducibility compatible with medium-throughput screening methods. The eTag technology was used to simultaneously measure the level of expression of four mRNAs—c-fos, c-jun, c-myc, and gapdh—in NGF-treated PC12 cells in a standard 96-well format. The experimental data shown here demonstrate the use of eTag technology as a new screening tool, which uniquely combines robustness, sensitivity, multiplexing capability, and direct measurement of mRNA without any sample preparation steps, such as RNA extraction/purification or a reverse transcription step.