FastLane Cell Probe Kit (200)

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Publication protocol

Total RNA was prepared from cells grown in 384-well plates or 96-well plates with FastLane Cell Probe kit or FastLane Cell Probe Multiplex kit (Qiagen). RNA lysates were used in real-time one-step RT-qPCR reactions performed on a Lightcycler 480 instrument (Roche). Gene expression values were calculated using the comparative delta delta Ct (-􏰀􏰀Ct) method, using housekeeper gene Ct values and control-treated Ct for normalization as described previously (20). FAM MGB Assay Probes were obtained from Thermo Fisher Applied Biosystems: KRAS (Hs00364284 g1), FOSL1 (Hs00759776 S1), 18S rRNA (4352655), RAB5C (Hs00904926 g1), RNASEH1 (Hs 007702360 s1), KIF1A (Hs00987720 m1), KIF5C (Hs00189672 m1), RAB5A (Hs00702360 s1) and RAB5B (Hs00951468 M1). For dual-colour RT-qPCR reactions, 18S rRNA VIC (4318839) was used. For polymerase chain reaction (PCR) analysis of ASO dose response treatment, curves were plotted and data were analysed using non- linear regression and curves were fitted using log (inhibitor) versus response, variable slope (four parameters) equation using GraphPad Prism, curves were constrained between 0 and 1.



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