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D Li, W Ma, F Sun, A Spector; The Confirmation of Expression Chip Analysis by Real-Time RT-PCR . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2366.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: This laboratory is interested in defining genes which are capable of protecting cells from oxidative stress. One approach is to analyze the gene expression of cell lines conditioned to resist such stress. While this can be done effectively by analysis with expression microchips, it is essential to have independent verification of the results. For this purpose, the laboratory is utilizing real-time reverse transcription polymerase chain reaction (RT-PCR). Methods: In this method, the total RNA, usually around 1 µg, is reverse transcripted producing the first strand cDNA. Real-time PCR is then conducted with specific primer pairs for the standards and the selected genes. SYBR green I is used to monitor PCR product. The dye binds non-specifically to only double stranded DNA, greatly enhancing its fluorescence which is detected at 530 nm with excitation at 470 nm. Upon melting, the fluorescence drops precipitously. Since the fluorescence is monitored at the end of each PCR cycle, it is possible to follow the increase in fluorescence as a function of cycle number. By using primers which are specific for a given DNA, the amplification of a given gene can be determined. Quantitation is possible since the internal standards are analyzed at the same time. Results: Prior microchip analysis defined a relatively small group of antioxidative genes in conditioned cell preparations which have significant change in their expression. This group included catalase, a number of GSH-S-transferases, NADPH menadione oxidoreductase, aldehyde oxidase 1, macrosialin and hephaestin. In order to find internal standards for real-time RT-PCR confirmation of these results, a number of classical housekeeping genes were examined further but found to be affected by the oxidative stress conditioning. The chip data was scanned to find housekeeping genes that had little or no variation under different conditions. Ribosomal protein L7 and ribosomal protein mitochondrial S24 as well as GAPDH were chosen. The latter standard, however, showed some variation at the enzyme activity level as well as small fluctuations in the chip data and, therefore, was not a primary reference. The results of these analyses will be reported. Conclusion: The verification of microchip data with real-time RT-PCR suggests that most results based on microchip analysis of gene expression indicating an = or ≷ 5 fold change are probably valid.
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