DDRT-PCR: Use of agarose gels for detection of amplified products

The present study focuses on the detection of differentially expressed gene s in migrating (healing) and nonmigrating (normal) corneal epithelium on ag arose gel using a modified procedure of differential display reverse transc riptase-polymerase chain reaction (DDRT-PCR). Rabbit corneal epithelial org an cultures were used to obtain nonmigrating and migrating samples. RNA was extracted using Trizol LS reagent. PCR was modified in order to allow dete ction of amplified products on 3% agarose gel with ethidium bromide stainin g. Products were also resolved on 6% denaturing polyacrylamide-urea gels an d observed by silver staining. Agarose gels showed two prominent bands that were heavily expressed in the 458 bp and 587 bp region of the nonmigrating samples. In addition light bands were visible in the region corresponding to 234 bp and 450 bp. In the migrating samples, two light bands were visibl e in the region of 267 bp and 300 bp. Eight amplicons, six in the nonmigrat ing corneal epithelial sample and two in the migrating corneal epithelial s amples, were also found to be differentially expressed when products were r un on 6% denaturing polyacrylamide-urea gels. Thus, DDRT-PCR products can b e detected on agarose gels and prove very helpful and economical in the ini tial studies of DDRT-PCR.