But:  To use microarray and qRT-PCR to identify changes in cell cycle genes expression in human corneal endothelial cells (CEC) from “in vivo”, post mortem, and organ cultured (OC) corneas, and also in confluent primary culture and immortalized CEC.

Materials and Methods:  Total RNAs were extracted “in vivo” corneas were obtained during penetrating grafts for keratoconus, immediately after trephination, post mortem corneas (body donation to Science) within 24H after death. OC corneas were stored for 15 days. Descemet was peeled off to avoid contamination with other cell types. Biotin-labeled cRNA probe was synthesized from RNAs extract and hybridized to the pretreated Oligo GEArray Human Cell Cycle Microarrays, which covered 112 key genes of cell cycle. Microarrays were performed in duplicate. Gene expression of “in vivo” CEC served as a reference. A difference >1.5 fold of the transcriptional level was considered significant. QRT-PCR were done on cyclin E1; cyclin D1; p16INK4a; p21CIP1; p27Kip1; Ki67 and Culin 1 to confirm the microarrays;

Results:  Compared to “in vivo”, CEC from OC corneas had 3 up regulated gene (CCNG1; CDK7; RBBP8) and 19 down regulated (CDC16; CDKN1B; MCM2; PCNA…), post mortem corneas had 3 up(CCNG1; CDK7; CKS2) genes and 14 down regulated (ATM; BAX; CDC16; CDKN1B…), primary culture had 1 up(CKS2) and 8 down regulated (BAX; CCNE1; CDK2; MCM2…), and the immortalized cell line had 16 up (ATM; CCNH; CKS2; MCM2…) and 2 down regulated genes (BAX; CDK5R2). QRT-PCR of the 5 aforementioned genes validated the Microarrays data.

Conclusion:  Microarrays are powerful tool to better understand the proliferative status of human CEC. It will help to choose the targets we need to alter in order to trigger CEC proliferation.