SIMPLIFIED RT PCR QUANTITATION OF GENE TRANSCRIPTS IN CULTURED NEUROBLASTOMA (SN49) AND MICROGLIAL (BV-2) CELLS USING CAPILLARY ELECTROPHORESIS AND LASER-INDUCED FLUORESCENCE

We developed a simplified protocol for sensitive quantitation of mRNA using polymerase chain reaction (PCR) amplification of cDNA made by re verse transcriptase (RT), as resolved with capillary electrophoresis ( CE) and detected with laser-induced fluorescence (LIF). The conditions required for adequate accuracy of the simplified version of the RT/PC R quantitation, in which a single concentration of external standard a nd amplification to within or near the plateau phase are used, were es tablished for assay of mRNAs expressed at high, moderate, and low abun dance. The mRNAs for the cytosolic enzyme, glyceraldehyde phosphate de hydrogenase (GAPDH) and the growth-associated protein GAP-43 in cultur ed SN49 neuroblastoma cells were used as target genes for high and mod erate levels of expression, respectively. Using cultured mouse microgl ial cells (BV-2), we demonstrated the utility of this RT/PCR/CE/LIF pr otocol to quantitate a low-abundance mRNA, encoding a form of nitric o xide synthase (i-NOS) induced by treatment with endotoxin. The appeara nce of i-NOS mRNA after endotoxin treatment of BV-2 cells was confirme d by Northern blot analysis and in situ hybridization histochemistry, and functional enzyme activity was followed by release of nitric oxide (as nitrite) into the medium. The many advantages of the 'single-poin t' RT/PCR/CE/LIF protocol for quantitating mRNAs of interest include: simplified protocol, elimination of the use of radiotracers, high sens itivity and precision, and semi-automation of the quantitation phase o f analysis.