DDRT-PCR: use of agarose gels for detection of amplified products.
The present study focuses on the detection of differentially expressed genes in migrating (healing) and nonmigrating (normal) corneal epithelium on agarose gel using a modified procedure of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). Rabbit corneal epithelial organ cultures were used to obtain nonmigrating and migrating samples. RNA was extracted using Trizol LS reagent. PCR was modified in order to allow detection of amplified products on 3% agarose gel with ethidium bromide staining. Products were also resolved on 6% denaturing polyacrylamide-urea gels and 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 visible in the region of 267 bp and 300 bp. Eight amplicons, six in the nonmigrating corneal epithelial sample and two in the migrating corneal epithelial samples, were also found to be differentially expressed when products were run on 6% denaturing polyacrylamide-urea gels. Thus, DDRT-PCR products can be detected on agarose gels and prove very helpful and economical in the initial studies of DDRT-PCR.
Ahmed
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Department of Biochemistry, University of Karachi, Karachi-75250, Pakistan. nikhat_ahmed14@yahoo.co.uk
Siddiqui
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Ahmed
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D000818:Animals; D046508:Culture Techniques; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019573:Epithelium, Corneal / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016622:Silver Staining; D014945:Wound Healing
D012333:RNA, Messenger