Differential display RT-PCR for identifying novel gene expression in the lung
The unique identity of each cell is the result of differential gene expression. A new strategy for differential cDNA screening introduced by Liang and Pardee utilizes anchored oligo-dT primers and random 5' oligonucleotide 10-mers to carry out polymerase chain reaction (PCR) on reverse-transcribed RNA from different cell populations. The amplified cDNAs are displayed on a standard sequencing gel as 100-500 bands per lane on the resulting autoradiograms and comparisons are drawn between the different cell populations. The major advantages of this method over previous differential screening approaches are its high sensitivity, the small amounts of tissue required, and the ability to carry out rapid mRNA analyses using total RNA and to test multiple tissues in parallel. Its limitations are the need for many primer combinations for adequate representation of mRNAs and the large number of bands displayed. A screening strategy should include multiple positive and negative control samples and Northern blots to identify cDNAs differentially expressed. This approach should facilitate the identification of many novel genes expressed in a variety of physiological and pathological conditions.