Journal of Chromatography B: Biomedical Sciences and Applications
Capillary electrophoresis methodology for identification of cancer related gene expression patterns of fluorescent differential display polymerase chain reaction
Introduction
Differential display PCR, originally described by Liang and Pardee [1], has been employed as a methodology for the study of altered gene expression events in a variety of cell types and tissues. In this technique, RNA from two or more samples is reverse transcribed using a 3′ anchored oligo-dT primer and PCR amplified using the same oligo-dT primer and a random arbitrary primer. Incorporation of radiolabeled nucleotides in the PCR reaction allows the resulting PCR products, representing a subset of the total mRNA, to be separated by polyacrylamide gel electrophoresis and visualized by autoradiography. Differential display has proven useful in the identification of genes involved in an array of biological processes including differentiation, hormonal regulation, apopotosis and carcinogenesis 2, 3, 4, 5, 6, 7, 8, 9, 10, 11.
The traditional isotopic methods for differential display are both time consuming and labor intensive. As a result, numerous technical modifications and improvements including nonisotopic and fluorescent methodologies have been reported 12, 13, 14, 15, 16, 17, 18, 19, 20. We have recently reported a fluorescent adaptation to the standard reaction that takes advantage of the specificity, selectivity and differential fluorescence of three dye labeled oligo-dT primers [21]. The advantages of this high throughput technique include decreased time and expense as compared to traditional isotopic methods and the ability to simultaneously screen and compare banding patterns from three different primer combinations. We have utilized this approach to identify expression events in a wide range of samples including bacteria, breast cancer cell lines, human cells and tumor samples.
In this report, we describe the use of the Perkin-Elmer ABI 310 Genetic Analyzer (capillary electrophoresis system) for the detection and identification of EST patterns generated using the fluorescent differential display technique. The ABI 310 uses a flowable polymer for uniform separation and automatically loads the samples. Using a human breast cancer cell line and a mouse mammary tumor model system, we have compared the patterns generated on both the ABI 377 and the ABI 310 instruments. The data generated on the ABI 310 was further analyzed using the ABI GeneScan and Genotyper software for semi-automated pattern recognition and comparison.
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Tissue culture and RNA isolation
MCF7 breast adenocarcinoma cells (ATCC HTB-22) were grown in a humidity controlled incubator at 37°C and 5% CO2 in Dulbeccos modified Eagles medium with 1 mM sodium pyruvate, 10 μg/ml bovine insulin and 10% fetal bovine serum. Total RNA was isolated from cell monolayers using Trizol LS (Life Technologies, Gaithersburg, MD, USA) according to the manufacturers protocol. Mouse mammary tumor samples A and B were homogenized and total RNA extracted as above. The isolated RNA was treated with
Results and discussion
Fluorescent differential display was performed using high quality RNA isolated from MCF7 human breast adenocarcinoma cell monolayers and from paired mouse mammary tumor samples A and B. As shown in Fig. 1, RNA was reverse transcribed using separate reactions for each of the three fluorescently labeled oligo-dT primers. These cDNAs were then PCR amplified with three different fluorescently labeled oligo-dTs and twenty-six different arbitrary decamers in duplicate. Appropriate experimental
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