Amplification ratios derived from rtqPCR using GAPDH 3′, midtranscript, and 5′ primer sets
| GAPDH amplification ratios | ||||||||
|---|---|---|---|---|---|---|---|---|
| 3′/mid 1st run | 3′/5′ 1st run | 3′/mid 2nd run | 3′/5′ 2nd run | |||||
| Ambion | Affy-mod | Ambion | Affy-mod | Ambion | Affy-mod | Ambion | Affy-mod | |
| 1, 10, and 100 ng uRNA and 10, 100, and 1000 BC-H1 cell RNA preparations were subjected to one or two rounds of amplification using the Ambion and modified Affymetrix (Affy-mod) protocols. We measured aRNA yields by rtqPCR targeting GAPDH with 3′, midtranscript, and 5′ primer sets. 3′ to midtranscript or 3′ to 5′ ratios were measured for each round, both protocols, and the various amounts of RNA. | ||||||||
| aRNA, amplified RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; rtqPCR, real time quantitative polymerase chain reaction; uRNA, universal RNA. | ||||||||
| RNA (ng) | ||||||||
| 1 | 2 | 2 | 3 | 2 | 11 | 3 | 31 | 8 |
| 10 | 3 | 1 | 5 | 2 | 5 | 3 | 10 | 8 |
| 100 | 2 | 2 | 2 | 2 | 8 | 2 | 14 | 8 |
| Cell numbers | ||||||||
| 10 | 5 | – | 7 | – | 30 | – | 102 | – |
| 100 | 11 | – | 76 | – | 8 | – | 21 | – |
| 1000 | 8 | 2 | 59 | 8 | 14 | 4 | 14 | 11 |