TABLE 2.
Effect of silencing and HSV-1 infection on mRNA half-life
| Infection | mRNA half-life (h) of:
|
|||
|---|---|---|---|---|
| EGFP
|
β-Actin
|
|||
| Silenceda | Controlb | Silenceda | Controlb | |
| Nonec | 2.8* (2.6-3.1) | 5.5 (5.2-5.7) | 21.7 (19.9-23.4) | 21.9 (20.1-23.1) |
| Mockd | 2.7* (2.6-3.0) | 5.4 (5.1-5.6) | 20.4 (19.8-21.1) | 21.2 (20.3-22.1) |
| Wild-type (KOS)d | 4.5** (4.3-4.6) | 4.1** (3.9-4.3) | 15.5** (15.0-16.1) | 15.4** (14.9-16.2) |
| Mocke | 2.8* (2.5-3.0) | 5.5 (5.3-5.8) | 20.6 (19.3-22.1) | 21.6 (20.1-22.8) |
| vhs-1e | 5.6** (5.3-5.8) | 5.4 (5.3-5.7) | 21.1 (19.8-22.6) | 20.5 (19.6-22.3) |
| 27-LacZe | 5.3** (5.2-5.6) | 5.7 (5.4-5.9) | 20.5 (18.9-22.1) | 20.5 (19.1-21.9) |
BHK cells were cotransfected with equimolar amounts of EGFP target-expressing and dsEGFP hairpin-expressing plasmids. The mRNA amounts (normalized to the 18S rRNA level) were determined from Northern blots probed first by hybridization with EGFP-specific sequences. Blots were stripped and subsequently hybridized to a β-actin-specific probe. Half-lives were estimated by fitting the data to an exponential decay function as described in Materials and Methods. Nonlinear regression analysis was performed, and the 95% confidence intervals for the estimated half-lives are shown in parentheses. A single asterisk indicates that the half-life estimates of silenced compared to control samples for each mRNA type were significantly different (P < 0.05). Double asterisks indicate that estimated half-lives of a particular mRNA type in infected samples were significantly different (P < 0.05) from those in mock-infected samples.
BHK cells were cotransfected with equimolar amounts of EGFP target-expressing and dslacZ hairpin-expressing plasmids. Half-lives were estimated and compared for differences as described in Materials and Methods and footnote a.
Cells were cotransfected with target- and hairpin-expressing plasmids as indicated, and Act D (5 μg/ml) was added 52 h later. RNA levels were determined at various times after the addition of Act D.
Cells were cotransfected with target- and hairpin-expressing plasmids as indicated, and 42 h later, cells were either mock infected or infected with wild-type (KOS) virus at an input MOI of 5 PFU/cell. Act D (5 μg/ml) was added 6 h after infection or mock infection, and mRNA levels were determined at various times thereafter.
Experiment was performed as described above except that cells were either mock infected or infected with the vhs-1 or 27-LacZ virus mutant (MOI of 5 PFU/cell).