Use of ~(33)P-Labeled Primer Increases the Sensitivity and Specificity of mRNA Differential Display

摘要

Liang and Paidee recently described a very useful technique for identifying differences in gene expression in subsets of RNAs which they called mRNA differential display (4). This method involves reverse transcription of mRNA with an anchored oligo-dTprimer and subsequent polymerase chain reaction (PCR) amplification of the cDNA in the presence of a second short primer of arbitrary sequence that anneals at different regions relative to the first primer. On using this method to study the effect of glucose on gene expression in rat pancreatic islets, we found that a significant proportion of PCR products were derived from amplification of far upstream regions of the mRNA rather than those proximal to the poly(A) site. In addition, many of the PCR products whose levels appeared to change when islets were cultured in high glucose concentrations were false positives and not derived from differentially expressed mRNAs. Sequencing of these false positive bands revealed the existence of the short arbitrary primer sequence or an unknown sequence at either end of the fragment and the absence of the anchored oligo-dT primer sequence, suggesting that unrelated sequences in far upstream regions of mRNA were amplified Here we describe a modified method of mRNA differential display that eliminates these false positives. We also describe the usefulness of using ~(33)P instead of ~(35)S for rapid detection of the positive signals without significant decrease in band resolution.