Table 4.
U6 expression in various vectorsa
| Name | Pol II enhancer | Size (kb) | pg RNAb (Run 1) | pg RNAb (Run 2) | Avg | Stdc |
|---|---|---|---|---|---|---|
| pGEM3ZF(+) U6 | None | 3.6 | 3.5 | 10.9 | 7.2± 5.2 | 100% c |
| NTC8485-U6 (SV40-)a | None | 2.0 | 15.8 | 13.7 | 14.8± 1.5 | 114% |
| NTC8485-U6a | SV40 | 2.2 | 9.6 | 13.3 | 11.5± 2.6 | 98% |
| NTC8485E-U6 (SV40-)a | CMV | 2.7 | 10.9 | 13.3 | 12.1±1.7 | 126% |
| NTC8485E-U6a | SV40+CMV | 2.8 | 7.1 | 12.5 | 9.8±3.8 | 106% |
| NTC8485E-U6 dimera | SV40+CMV | 5.6 | 5.4 | 16.3 | 10.9±7.7 | 235%d |
| NTC7485-U6 | SV40 | 3.0 | 8.2 | 9.9 | 9.1±1.2 | 105% |
| NTC7485E-U6 | SV40+CMV | 3.6 | 7.9 | 9.4 | 8.7±1.1 | 121% |
| pDNAVACC EGFP-U6 | CMV EPe | 4.3 | 4.2 | 5.8 | 5.0±1.1 | 83% |
| pDNAVACC EGFP-U6-VAI | CMV EPe | 4.5 | 4.1 | 4.3 | 4.2±0.1 | 73% |
| pMLV GFP-neo U6 shRNA | CMV EPe SV40 E | 7.1 | 3.8 | 3.6 | 3.7±0.1 | 101% |
| pDNAVACC EGFP (negative control) | CMV EPe | 3.8 | 0 | 0 | 0 | 0 |
a
Vectors expressed a 100 bp ssRNA (eRNA18) for RT-PCR quantification. Vector sizes are 100 bp larger than the corresponding shRNA vectors from Table 3. Transfections were performed in duplicate in 24 well plates, using two wells per transfection (4 wells per experiment).
b
pg eRNA18 target/100 ng total RNA isolated 24 h post transfection.
c
Standardized mU6 expression compared to pGEM3F(+) U6 vector (avg pg RNA/7.2 × vector size (kb)/3.6 × 100%).
d
Dimer contained two copies of eRNA18 per plasmid.
e
Vector contained CMV promoter and RNA Pol II expressed transgene.