NCCIT cells have previously been shown to have an STI571 approximately 4 fold higher IC50 to cisplatin, com pared to NTERA 2. We utilized a quantitative RT PCR based platform for detection of almost all currently known human micro RNA species on these 6 cell lines. Our approach was designed to charac terize the role of micro RNAs on the presumably multi factorial phenomenon of acquired cisplatin resistance in germ cell tumors. Methods 1 Cell lines Both chemo sensitive paternal 2102EP and NCCIT cells as well as their cisplatin resistant sublines were cultured in DMEM F12 medium containing 10% fetal calf serum. NTERA 2 and the cis platin resistant subline were cultured in DMEM medium supplemented with Glutamax I containing 10% fetal calf serum. Cells were incubated at 37 C in a humidi fied atmosphere with 5% CO2 and were passaged every 3 4 days.
After trypsination of cultured cells from Inhibitors,Modulators,Libraries steady state conditions and at comparable cell density, aliquots containing 10 106 cells were transferred into 1 ml RNA later solution and stored at 20 C. Three aliquots per cell line originating from different cell passages Inhibitors,Modulators,Libraries were processed and stored as described above. 2 Induction of cisplatin resistance The cisplatin resistant sublines NTERA 2 R, 2102EP R and NCCIT R were generated by intermittent exposure of the parental cell lines to cisplatin over a time period of 18 month, starting with the respective IC10 dose of each parental cell line. At reaching 50% cell kill, the addition of cisplatin was interrupted and cells were allowed to recover over three passages.
Analyses pre sented here were Inhibitors,Modulators,Libraries performed after maintaining the resis tant sublines for at least 1 week in cisplatin free medium to allow for an adequate wash out period. Results of cytotoxicity experiments of all six cell lines using the MTT assay 2,5 Diphenyltetrazolium Bromide have been published The experiments were performed in 3 completely inde pendent replicates for every cell line including genera tion of resistant cell lines, cell culture, RNA isolation and RTQ PCR measurements. 3 RNA Isolation Cells were transported on dry ice in RNA Inhibitors,Modulators,Libraries later solution and stored at 80 C until use. After thawing the cells, they were disrupted using a denaturing lysis buffer, and RNA was isolated by com bining organic extraction followed by immobilization of RNA on glass fiber filters employ ing the mirVana miRNA Isolation Kit in order Inhibitors,Modulators,Libraries to purify total RNA including small RNA species.
Remaining DNA was digested on glass fiber filters, and after per forming quality controls, RNA was stored at 80 C until analysis by RTQ PCR. To control the quality and purity of isolated total RNA including small RNA, we performed spectral photometry, agarose click this gel electrophoresis, and PCR. Only high quality total RNA including small RNA species was used for further experiments.
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