Measurement of single-cell gene expression using capillary electrophoresis.
Capillary electrophoresis with laser-induced fluorescence detection was used to monitor gene expression in individual mammalian cells using the reverse transcriptasepolymerase chain reaction. Specifically, beta-actin expression in single LNCaP (prostate cancer) cells was measured. A sieving matrix containing hydroxypropyl methyl cellulose was used to effect size-based separation. Ethidium bromide fluorescence of the product DNA was used as the detection scheme and yielded excellent sensitivity. The beta-actin product, resulting from an individual cell lysed by a freeze-thaw method, gave an average signal-to-noise ratio (S/N) of 77+/-27 (n = 2). Chemical lysis of a single cell, using a dilute solution of SDS, gave a S/N of 26+/-2 (n = 2), roughly 3-fold lower than for freeze-thaw lysis. An initial detection limit (not considering fully optimized conditions) was calculated from an amplified cDNA standard to correspond to a concentration of approximately 133 starting molecules/nL (of beta-actin mRNA).
Zabzdyr
J L
JL
Department of Chemistry, University of California, Riverside 92521, USA.
Lillard
S J
SJ
D019075:Electrophoresis, Capillary; D015870:Gene Expression; D006801:Humans; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D011506:Proteins