Table 2.
Target RNA Concentrations (Copies/mL) |
Target RNA Concentrations in Reaction (Copies/μL) (1000 μL Reaction Mixtures) |
Theoretical Calculation (N = 96) |
Actual Results | |||||
---|---|---|---|---|---|---|---|---|
λ | Np | Nn/N | Np | N | λ | C (Copies/μL) | ||
10 | 0.001 | 0.0104 | 1 | 0.9896 | 2 | 96 | 0.0208 | 0.0019 |
102 | 0.01 | 0.1040 | 10 | 0.9019 | 8 | 96 | 0.0870 | 0.0084 |
103 | 0.1 | 1.0416 | 62 | 0.3535 | 58 | 96 | 0.9268 | 0.0885 |
104 | 1 | 10.4167 | 96 | 0 | 96 | 96 | ≥9.6 | ≥0.96 |
N: the total number of wells. Np: number of positive wells. Nn: number of negative wells. λ: copies per well = −ln(Nn/N) = Copies in the reaction/Total wells = Concentration in reaction (Copies/μL) × V (volume of each well). V = Reaction volume/N = 10.4 μL. P(0) = Nn/N = e−λ, λ = −ln(Nn/N) = Concentration in reaction × V. Concentration in reaction = [−ln(N/N)]/V. Target RNA concentration = 10 × CCopies/μL = 104 × CCopies/mL.