A qualitative look at multiplex gene expression of single cells using capillary electrophoresis.
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.
Zabzdyr
Jennifer L
JL
Department of Chemistry, University of California, Riverside, CA, USA.
Lillard
Sheri J
SJ
D000199:Actins / Q000235:genetics; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000235:genetics; D005260:Female; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D007834:Lasers; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D047628:Estrogen Receptor alpha; D012333:RNA, Messenger