User manual

56 | Droplet Digital
™
PCR Applications Guide
In the example given, the measurement error (CV) is introduced at the time of subsampling
(at the time of the blood draw), not by the measurement system. Poisson statistics
dictate that if N target molecules are measured in a sample, the standard deviation of the
measurement is roughly the square root of N. In this example, if 100 copies of virus are
present in the sample, the standard deviation of the measurement is 10 and the CV is
10/100 = 10%. This means that we can accurately quantify, to ±10%, 100 or more copies
of virus in 100,000 PBMCs. No detection system can do better than this theoretical limit.
Factors that Impact RSD Calculations
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False-positive rate — the false-positive rate must be measured and considered when
designing experiments to achieve very low LoD or accurate quantification
■
Sample integrity and preparation — it may be useful to spike a PCR control into
the original sample and use a second assay in the same well to measure its level.
This enables you to control for PCR inhibitor and sample preparation failures
■
Pipetting accuracy in dilutions — errors introduced by pipetting may contribute to the
overall accuracy of the measurement
References
Benlloch S et al. (2006). Detection of BRAF V600E mutation in colorectal cancer:
Comparison of automatic sequencing and real-time chemistry methodology.
J Mol Diagn 8, 540–543.
Kwok S and Higuchi R (1989). Avoiding false positives with PCR. Nature 339, 237–238.
Scott LM (2011). The JAK2 exon 12 mutations: A comprehensive review.
Am J Hematol 86, 668–676.
Whitehall V et al. (2009). A multicenter blinded study to evaluate KRAS mutation
testing methodologies in the clinical setting. J Mol Diagn 11, 543–552.
Rare Mutation and Sequence Detection