Table 2. Effect of external standards mixture (ESM) on quality control.
| Without ESM correction | ||||||||||||
| ACTB | MYC | |||||||||||
| Sample | Day | ISM | NT Cq | IS Cq | Raw ΔCq | Molecules/Assay | NT Cq | IS Cq | Raw ΔCq | Molecules/Assay | MYC/106 ACTB | |
| SM8 | #1 | D(−12/−14) | 11.7 | 13.0 | −1.3 | 1.5E+06 | 16.7 | 18.5 | −1.8 | 2.1E+04 | 1.4E+04 | |
| #2 | D(−12/−14) | 11.8 | 11.3 | 0.5 | 4.3E+05 | 19.7 | 16.4 | 3.3 | 6.0E+02 | 1.4E+03 | ||
| E(−12/−15) | 11.7 | 11.0 | 0.7 | 3.7E+05 | 18.7 | 19.0 | −0.2 | 7.1E+02 | 1.9E+03 | |||
| #3 | D(−12/−14) | 11.6 | 10.8 | 0.8 | 3.4E+05 | 19.8 | 16.9 | 2.9 | 8.1E+02 | 2.4E+03 | ||
| E(−12/−15) | 12.2 | 11.2 | 1.0 | 3.0E+05 | 19.8 | 20.4 | −0.6 | 9.0E+02 | 3.0E+03 | |||
| #4 | D(−12/−14) | 18.0 | 16.6 | 1.4 | 2.3E+05 | 26.2 | 24.3 | 1.9 | 1.6E+03 | 7.0E+03 | ||
| #5 | D(−12/−14) | 12.1 | 13.6 | −1.5 | 1.7E+06 | 20.2 | 19.7 | 0.5 | 4.3E+03 | 2.5E+03 | ||
| CV | 0.90 | 1.75 | 0.99 | |||||||||
| With ESM correction | ||||||||||||
| SM8 | #1 | D(−12/−14) | −1.3 | −2.0 | 0.7 | 3.8E+05 | −1.8 | −3.0 | 1.2 | 2.5E+03 | 6.6E+03 | |
| #2 | D(−12/−14) | 0.5 | 0.0 | 0.4 | 4.5E+05 | 3.3 | 2.3 | 1.1 | 2.9E+03 | 6.5E+03 | ||
| E(−12/−15) | 0.7 | 0.0 | 0.6 | 3.8E+05 | −0.2 | 2.3 | −2.5 | 3.4E+03 | 8.8E+03 | |||
| #3 | D(−12/−14) | 0.8 | −0.6 | 1.4 | 2.3E+05 | 2.9 | 1.7 | 1.2 | 2.7E+03 | 1.2E+04 | ||
| E(−12/−15) | 1.0 | −0.6 | 1.6 | 2.0E+05 | −0.6 | 1.7 | −2.3 | 2.9E+03 | 1.5E+04 | |||
| #4 | D(−12/−14) | 1.4 | 0.5 | 0.9 | 3.3E+05 | 1.9 | 0.9 | 1.1 | 2.9E+03 | 8.8E+03 | ||
| #5 | D(−12/−14) | −1.5 | −2.1 | 0.6 | 3.9E+05 | 0.5 | −0.5 | 1.0 | 3.0E+03 | 7.7E+03 | ||
| CV | 0.27 | 0.09 | 0.32 | |||||||||
Inter-day experimental variation without and with ESM.
Note: SM8: surgically removed, formalin-fixed, paraffin-embedded (FFPE) sample 8. ISM D (−12/−14) contains ACTB IS 10−12 M/target gene IS 10−14 M that corresponds to ACTB IS 600000/target gene IS 6000 molecules. ISM E (−12/−15) contains ACTB IS 600000/target gene IS 600 molecules. Cq: quantification cycle. ΔCq: quantification cycle difference of NT and IS: NT Cq - IS Cq. CV: coefficient of variation. NT: native template. IS: internal standard. ISM: internal standards mixture.