Cells treated with inhibitors were lysed with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) for total RNA isolation. Subsequently, RNA extract was treated with TURBO DNA-free™ Kit (Ambion, Carlsbad, USA) in order to get rid of DNA contamination. The treated samples were thereafter subjected to cDNA synthesis using random hexamers (Amersham) at 65°C for 10 min and then at 37°C for 1 h. Quantitative RT-PCR was performed for gene specific transcript using primers specific for mouse were: caspase-8-Fwd, 5′-GAGGATGCCCAGAAGCTA-3′; caspase-8-Rev, 5′-CCG CAGCTCTCTCACCAT-3; caspase-3-Fwd, 5′-AATGTCATCTCGCTC TTGGT-3′; caspase-3-Rev, 5′-GCTTAGAATCACAC ACAC-3′; BAX-Fwd, 5′-CACCAGCTCTGAACAGAT-3′; BAX-Rev, 5′-CCAGTTCATCTCCAATTCG-3′. The reactions were carried out in an ABI Prism 7500 sequence detection system (Applied Biosystems, CA, USA). The qRT-PCR results were analyzed using the iCycler Thermal Cycler Software (Applied Biosystems, CA, USA) and normalized with those from 18S rRNA internal control (Das et al., 2009 (link)).
Free full text: Click here
