Capillary electrophoresis methodology for identification of cancer related gene expression patterns of fluorescent differential display polymerase chain reaction.
The mRNA differential display technique is a method for molecular survey and analysis of differential gene expression in eukaryotic cells and tissues. We have previously described the use of ABI Prism fluorescent technology to specifically amplify expressed sequence tags (ESTs) from several different biological paradigms. High throughput, fluorescent differential display performed on an automated sequencer (ABI 377) has proven to have significant cost cutting and time saving attributes compared to that of the radioactive differential display. Additionally, fluorescent tagged mRNA specific reverse transcription and PCR decreases the number of the inherent artifacts associated with radioactive differential display. We report here the application of a capillary electrophoresis system (ABI 310) to the identification of fluorescent differential display generated EST patterns. RNA samples from human and animal breast cancer paradigms were exposed to this technique and analyzed by the ABI 310 and the ABI 377. GeneScan and Genotyper software applications were used for rapid and semi-automated characterization of fluorescently labeled ESTs. Automated sample loading and uniform sample electrophoresis are among the main advantages of this system which significantly increase the precision and reproducibility of fluorescent differential display.
George
K S
KS
SRA Technologies, Rockville, MD 20850, USA.
Zhao
X
X
Gallahan
D
D
Shirkey
A
A
Zareh
A
A
Esmaeli-Azad
B
B
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000235:genetics / Q000473:pathology; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012334:RNA, Neoplasm / Q000032:analysis*; D015203:Reproducibility of Results; D012984:Software; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D012334:RNA, Neoplasm