The involvement of survival signaling pathways in rubella-virus induced apoptosis

Cooray, Samantha; Jin, Li; Best, Jennifer M

Total RNA was extracted from 100 µl tissue culture supernatants, collected at indicated times p.i., using a silicaguanidinium isothiocyanate method [44] . Prior to reverse transcription, RV RNA was heated to 95°C for 1 minute and kept on ice. RNA was transcribed to cDNA using Superscript III RNase Hreverse transcriptase (Invitrogen, UK). Reverse transcription was performed in 20 µl reaction volumes containing 200 U enzyme, 10 µl sample RNA, 0.5 mM of each dNTP, and 5 pmoles external reverse primer (5'-CCTGTACGTGGGGCCTTTAA-3'). RNA bound to cDNA in RNA-DNA hybrids was removed by incubation of the cDNA with RNase H (Roche Diagnostics, UK) for 20 minutes at 37°C. PCR amplification was carried out using a GC-Rich PCR System (Roche Diagnostics, UK). In the PCR reaction 10 µl cDNA was added to 40 µl of PCR reaction mix to give final concentrations of 1X GC-Rich PCR buffer, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 0.5 M GC-rich resolution solution™, 0.5 pmole of forward and reverse primers (5'-TAGGAGGTGCCGCCATATCA-3' and 5'-CCTGTACGTGGGGCCTTTAA-3' respectively), and 2U Taq polymerase and a mixture of proof-reading polymerases. The cycling conditions, as recommended by the manufacturer were: 95°C for 3 minutes followed by 10 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute; and 25 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute (plus an additional 5 seconds per cycle), and a final extension of 72°C for 7 minutes. Amplified capsid product (1053 b.p.) was electrophoretically separated on 1% Tris-Borate (TBE) agarose gels, stained with ethidium bromide solution (5 mg/ml) and visualized under UV light.