Table 3.
siRNA knockdowns and their effects on cells
| siRNA | Cell no., 106 (%)a | Proliferation rate (%)b | Plating efficiency (%)c | HPRT mRNA (fold)d | % Target mRNAe |
|---|---|---|---|---|---|
| Vimentin | 4.6 (100) | 0.82 (100) | 100 | 23 | |
| TOP1-1 | 2.3 (50) | 0.66 (81) | 98 | 31 | 29 |
| TOP1-2 | 2.4 (52) | 0.67 (82) | 87 | 25 | 31 |
| TDP1-1 | 1.7 (36) | 0.58 (71) | 111 | 30 | 34 |
| TDP1-2 | 1.6 (35) | 0.57 (70) | 106 | 32 | 37 |
| XRCC1-1 | 4.7 (103) | 0.83 (101) | 108 | 33 | 24 |
| XRCC1-2 | 4.9 (106) | 0.84 (102) | 109 | 21 | 28 |
| PARP1-1 | 3.5 (75) | 0.76 (93) | 61 | ND |
Cell number refers to the total number of cells present per plate after 6 days of siRNA treatment. Each plate began with an initial cell population of 150,000 cells. Treatment with vimentin siRNA was used as the reference and was defined as 100%.
The proliferation rate is the number of cell doublings per day required to increase the cell population at the beginning of the siRNA treatment (150,000 cells per plate) to the number present at the end of the 6-day treatment. For vimentin siRNA treatment, for example, the initial population of cells increased to 4.6 × 106 cells after 6 days, a 31-fold increase, which corresponds to 4.9 population doublings per 6 days, or 0.82 doublings per day. The proliferation rate during treatment with vimentin siRNA was used as the reference and was defined as 100%.
The plating efficiency was measured at the time cells were replated for selection for HPRT− cells. It is the percentage of colonies that formed when 200 cells were plated in nonselective medium at the time of plating. The absolute plating efficiency for vimentin siRNA-treated cells was 76%. This plating efficiency was used as the reference and was defined as 100%.
Induced HPRT mRNA levels in cells treated with specific siRNAs in the presence of doxycycline are expressed relative to the amount of HPRT mRNA in cells treated with vimentin siRNA in the absence of induction by doxycycline. In all cases the mRNA was first normalized to the β-actin mRNA in the same sample. HPRT mRNA levels were not determined (ND) in the samples treated with PARP1 siRNA.
The target mRNA is the amount of target gene mRNA present in cells treated with the specific siRNA relative to that present in cells treated with vimentin siRNA. For example, the amount of TOP1 mRNA present after treatment with TOP1-2 siRNA was 31% of the amount of TOP1 mRNA present in the vimentin siRNA-treated control. In all cases, the mRNA was first normalized to the β-actin mRNA in the same sample.