Figure 3

Inhibition of expression occurs post-transcriptionally and does not affect mRNA transport and stability. A) Total RNA was extracted from HEK 293T cells transfected with Mtv-1, HBRV, and SM gag constructs. The gag and gapdh mRNAs were reverse transcribed into cDNA using an Oligo-dT primer and quantified by Real-Time PCR. The relative abundance of gag mRNA to gapdh is calculated as the reverse of the ratio of gag CT to gapdh CT value. B) Transfected HEK 293T cells were separated into nuclear and cytoplasmic fractions. The mRNA was extracted from each fraction, and gag and gapdh mRNAs were quantified by reverse transcription Real-Time PCR. The level of mRNA transport from the nucleus to the cytoplasm was quantified as the ratio of nuclear and cytoplasmic gag mRNA relative to gapdh. C) Cells were treated with actinomycin D, and RNA was extracted from the cells at 0, 2, 4 and 6 hrs post-treatment and quantified by reverse transcription Real-Time PCR. The relative abundance of gag mRNA to gapdh was calculated at each time point. Data are the average of at least three independent experiments ± SD.