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 reverse 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/PCR quantitation, in which a single concentration of external standard and amplification to within or near the plateau phase are used, were established for assay of mRNAs expressed at high, moderate, and low abundance. The mRNAs for the cytosolic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH) and the growth-associated protein GAP-43 in cultured SN49 neuroblastoma cells were used as target genes for high and moderate levels of expression, respectively. Using cultured mouse microglial cells (BV-2), we demonstrated the utility of this RT/PCR/CE/LIF protocol to quantitate a low-abundance mRNA, encoding a form of nitric oxide synthase (i-NOS) induced by treatment with endotoxin. The appearance of i-NOS mRNA after endotoxin treatment of BV-2 cells was confirmed 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-point' RT/PCR/CE/LIF protocol for quantitating mRNAs of interest include: simplified protocol, elimination of the use of radiotracers, high sensitivity and precision, and semi-automation of the quantitation phase of analysis.
Personett
D A
DA
Mayo Clinic Jacksonville, Alzheimer Research Group, FL 32224, USA.
Chouinard
M
M
Sugaya
K
K
McKinney
M
M
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001932:Brain Neoplasms / Q000378:metabolism*; D018076:DNA, Complementary / Q000032:analysis / Q000378:metabolism; D019075:Electrophoresis, Capillary; D017403:In Situ Hybridization; D007834:Lasers; D051379:Mice; D017628:Microglia / Q000378:metabolism*; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase