Table 3. Titration of IC using ITR2 or target sequences specific to sample.
| Sample | Template | Run 1 (vg/ml) | Run 2 (vg/ml) | Run 3 (vg/ml) | Average (vg/ml) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| IC1: AAV2/5-GFP | ITR2 psub201/HindIII |
7.53 ± 1.84 E+13 |
6.66 ± 2.02 E+13 |
1.26 ± 0.46 E+14 | 8.93 ± 1.44 E+13 (P = 0.06) | ||||||
| Free ITR psub201/PvuII |
8.29 ± 1.88 E+12 |
1.57 ± 0.31 E+13 |
2.77 ± 0.54 E+13 | 1.72 ± 0.91 E+13 (N.S) | |||||||
| Target: GFP pFBGR |
1.64 ± 0.15 E+13 |
2.28 ± 0.21 E+13 |
2.37 ± 0.28 E+13 | 2.10 ± 0.21 E+13 | |||||||
| IC2: AAV2/6-GFP | ITR2 psub201/HindIII |
1.40 ± 0.55 E+14 |
1.43 ± 0.26 E+14 |
1.08 ± 0.54 E+14 | 1.30 ± 0.45 E+14 (P < 0.05) | ||||||
| Free ITR psub201/PvuII |
1.68 ± 0.25 E+13 |
2.96 ± 0.54 E+13 |
2.90 ± 0.77 E+13 | 2.51 ± 0.52 E+13 (N.S) | |||||||
| Target: GFP pFBGR |
2.14 ± 0.31 E+13 |
2.12 ± 0.25 E+13 |
1.93 ± 0.27 E+13 | 2.06 ± 0.28 E+13 | |||||||
| Template |
Run 1 |
Run 2 |
Run 3 |
||||||||
| r-squared | Slope | Efficiency | r-squared | Slope | Efficiency | r-squared | Slope | Efficiency | |||
| ITR2 psub201/HindIII | 99.8% | -3.04 | 107.0% | 98.2% | -3.03 | 106.9% | 99.9% | -3.28 | 96.3% | ||
| Free ITR psub201/PvuII | 99.2% | -3.33 | 99.9% | 99.1% | -3.42 | 98.0% | 100.9% | -3.51 | 96.3% | ||
| Target: GFP pFBGR | 99.9% | -3.63 | 94.4% | 99.8% | -3.65 | 94.0% | 99.9% | -3.54 | 95.8% | ||
ITR2 qPCR was performed using linearized plasmid (psub201/HindIII) or double digested plasmid with free ends at ITR sequences (psub201/PvuII) as standard curve. The standard deviations listed for Internal Controls in each run is from different dilutions of the same sample. P values were calculated between ITR2 and free ITR versus GFP and considered statistically significant when P < 0.05.
AAV, adeno-associated virus; GFP, green fluorescent protein; IC, internal controls; ITR, inverted terminal repeats; N.S., nonsignificant; qPCR, quantitative polymerase chain reaction.