Differential mRNA display analysis

We found that it is necessary to run several sets of differential display primers prior to an analysis of the distribution of differential display bands. This allows for a comparison between different independent reactions using different PCR primers to assess the quality of individual cDNA samples and discriminate between sample-to-sample variability and potential positive bands that are consistently found in different repUcates. The presence or absence of a specific band in lanes corresponding to independent experimental samples indicates a reproducible difference in the relative amount of cDNA in a given sample, which should reflect differences in mRNA levels. However, the interpretation of the differential display results is not always straightforward. For example, a thick band can reflect quantitative differences in the initial concentration of a specific cDNA between samples or can represent comigration of two bands. Replication of the PCR reactions for samples that have differences in banding pattern will eliminate a significant number of false positive differential display differences. Also, in some cases, it may be informative to alter the electrophoresis conditions to maximize resolution of a band of interest prior to isolation, reamplification, and further analysis of potential positive bands. 

Various methods are available for detecting and quantifying gene-expression levels, including northern blots, differential display, polymerase chain reaction after reverse transcription of RNA, and serial analysis of gene expression. These techniques are used primarily to measure the expression levels of specific genes or to screen for significant differences in mRNA abundance. 

Zimmermann, J. W. and Schultz, R. M. (1994) Analysis of gene expression in the preimplantation mouse embryo use of mRNA differential display. Proc. Natl. Acad. Sci. USA. 91,5456-5460. 

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