HeLa Cell Fractionation and mRNA Analysis

HeLa cells were transfected as described above and were described earlier [5 (link)]. For cell fractionation studies, the nuclear and cytoplasmic fractions were prepared as previously described prior to western analysis [15 (link),65 (link)] and performed at least twice for each type of experiment. RNA was purified using TriZol and Trizol LS. 1 µg of total RNA was treated with DNAse I Amplification Grade (Life Technologies) and first strand cDNA synthesis was performed using Superscript II RT (Life Technologies). 2 µL of total cDNA was taken and PCR amplified for gapdh mRNA and vRNA using the primers described in Abrahamyan etal. [61 (link)]. mCherry mRNA was amplified using a specific primer pair (Forward primer, 5'-TGG AGG GCT CCG TGA ACG GCC and reverse primer 5'-TAG GCG CCG GGC AGC TGC ACG) to generate a RT-PCR product of about 483 bp. Signal intensities of PCR signals (cycle 25–30) were quantitated exactly as described above [61 (link),62 (link)] using ethidium bromide stained DNA signals captured in-gel. All calculations were performed by taking into account the gapdh mRNA signal intensities.