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A qualitative look at multiplex gene expression of single cells using capillary electrophoresis
Author(s) -
Zabzdyr Jennifer L.,
Lillard Sheri J.
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200406126
Subject(s) - ethidium bromide , capillary electrophoresis , microbiology and biotechnology , multiplex , chemistry , dna , bovine serum albumin , gel electrophoresis , multiplex polymerase chain reaction , fluorescence , electrophoresis , polymerase chain reaction , chromatography , biology , gene , biochemistry , genetics , physics , quantum mechanics
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 α (ERα) and β‐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 ERα and β‐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 β‐actin, whereas the DNA carrier allowed co‐amplification of both ERα and β‐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.