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

[摘要] The present study focuses on the detection of differentially expressedgenes in migrating (healing) and nonmigrating (normal) cornealepithelium on agarose gel using a modified procedure of differentialdisplay reverse transcriptase-polymerase chain reaction (DDRT-PCR).Rabbit corneal epithelial organ cultures were used to obtainnonmigrating and migrating samples. RNA was extracted using Trizol LSreagent. PCR was modified in order to allow detection of amplifiedproducts on 3% agarose gel with ethidium bromide staining. Productswere also resolved on 6% denaturing polyacrylamide-urea gels andobserved by silver staining. Agarose gels showed two prominent bandsthat were heavily expressed in the 458 bp and 587 bp region of thenonmigrating samples. In addition light bands were visible in theregion corresponding to 234 bp and 450 bp. In the migrating samples, twolight bands were visible in the region of 267 bp and 300 bp. Eightamplicons, six in the nonmigrating corneal epithelial sample and two inthe migrating corneal epithelial samples, were also found to bedifferentially expressed when products were run on 6% denaturingpolyacrylamide-urea gels. Thus, DDRT-PCR products can be detected onagarose gels and prove very helpful and economical in the initialstudies of DDRT-PCR.

[学科分类] 生物化学/生物物理