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).

No abstract

No abstract

No abstract

We demonstrate the first use of capillary electrophoresis with laser-induced fluorescence (CE-LIF) for the qualitative analysis of single-cell multiplex products of the reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of both estrogen receptor alpha (ERalpha) and beta-actin in individual MCF-7 cells was monitored using a one-pot reaction. Reverse transcription and a single round of touch-down PCR, performed in a multiplex format, were used to generate fragment sizes of 318 bp and 838 bp, for ERalpha and beta-actin, respectively. A replaceable hydroxypropylmethylcellulose sieving matrix was used to effect a size-based separation of ethidium bromide-bound DNA. As titration of RT-PCR reaction components did not appreciably influence multiplex product generation, the use of additives, including bovine serum albumin (BSA) and herring sperm DNA, was explored. The addition of BSA to the RT-PCR mixture only resulted in efficient amplification of beta-actin, whereas the DNA carrier allowed co-amplification of both ERalpha and beta-actin. Furthermore, the sensitivity of our CE-LIF method eliminated the need for a second round of nested PCR, typically required when RT-PCR products are analyzed using gel electrophoresis.