To study DRPLA gene expression in human corneal endothelial cells, reverse transcriptase–polymerase chain reaction (RT-PCR) analysis was performed. We produced complementary DNA (cDNA) of the human corneal endothelial cell from human corneal endothelial cells, which were isolated from healthy donor cornea (obtained from Rocky Mountain Lions Eye Bank, Denver, Colo) as described previously.5 Total RNA was first isolated from human corneal endothelial cells using a monophasic solution of phenol and guanidine isothiocyanate (Isogen; Nippon Gene, Toyama, Japan), then treated with RNase-free DNase I (Stratagene, La Jolla, Calif) for 30 minutes. Human brain cDNA was purchased (Maxim Biotech, Inc, San Francisco, Calif), and amplification of the 557-base pair fragment of the DRPLA gene, which is designed to amplify exons 9 to 10, was performed using the 5′ primer 5′-AGGAGGACTACTACAGTCAC-3′ and 3′ primer 5′-TGGTTTTGGTTGGGATGTTT-3′. For the negative control of corneal endothelial cells, PCR was performed in the absence of RT. The PCR buffer contained 1.5mM magnesium chloride with 0.2mM of each deoxynucleotide triphosphate, 2µM of each primer, and 2.5 U of Taq polymerase (Takara Shuzo Co, Ltd, Shiga, Japan). The PCR analysis consisted of 1 cycle of 5 minutes at 94°C; 35 cycles of 1 minute at 94°C, 1 minute at 60°C, and 2 minutes at 72°C; and 1 cycle of 7 minutes at 72°C in a PCR system (GeneAmp System 2400; Perkin Elmer, Foster City, Calif). The PCR products were separated by means of electrophoresis through an 8% polyacrylamide gel and visualized using ethidium bromide under UV light.