Differential display using one-base anchored oligo-dT primers

Differential display was developed as a method to identify and analyze altered gene expression at the mRNA level in any eukaryotic cell. A similar method utilizing a single arbitrary primer to produce RNA finger-printing was developed independently and used for the same purpose as differential display. Despite the wide use of differential display in the analysis of altered gene expression, two major obstacles remained with the method. One is the redundancy and under-representation of certain mRNA species and the other is a rather high number of 'false positives' that cannot be confirmed by Northern blot analysis. To this end, various efforts have been made to optimize and streamline the method. While the problem could, to some extent, be attributed to the extrinsic factors such as pipetting errors, the integrity of RNA samples, contamination of RNA with chromosomal DNA or even bad PCR tubes, the intrinsic factors with the current scheme of the method have not yet been critically addressed. This work describes the use, in differential display, of three one-base anchored oligo-dT primers which provide excellent selectivity in subdividing mRNA into three populations. In comparison with two-base anchored oligo-dT primers, the use of one base-anchored oligo-dT primers further cuts down the number of reverse transcription reactions needed for each RNA sample, minimizes the redundancy and under-representation of certain RNA species due to the degeneracy of the primers. The introduction of a restriction site at the 5' ends on both the anchored and arbitrary primers makes the primers longer and more efficient in cDNA amplification while allowing the cloned cDNA to be more readily manipulated.