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Fig. 9 Minimum number of microbeads required for a stable ligand value. Microbeads were
either loaded with a capture probe for MLC-2v-cap or an artificial sequence designated aCS-cap.
Microbeads with different capture probe densities were used (m''low'', d ''medium'', and j
''high'' surface probe density). Vertical dashed black line is the cut-off for a stable ligand value.
For (a) MLC-2v-cap and (b) aCS-cap the number of microbeads were plotted against their
refMFI. Error bars represent the standard deviations. Quencher oligonucleotides for (c) MLC-2v-
cap and (d) aCS-cap were given to the samples and the hybridization efficiency ''H [%]''
calculated as Eq. 3 , where H 0 is the non-quenched and H 1 the quenched state in refMFI,
respectively. Microbeads with a medium (not shown) and high (j) capture probe density reported
higher variability at low (\80) microbead numbers which was not the case for microbeads with a
low (m) capture probe density
3.3 Determination of Minimum Number of Microbeads Per Assay
The universal advantage of microbeads is their n-fold redundancy at a desired
number in a single reaction environment, e.g., a well of a microplate. In theory,
statistical measures, including mean and median, can be provided at high data
quality and reproducibility. In reality, however, there is a limitation for the min-
imum number of features, mainly attributed to the variance within one microbead
population (see Rödiger et al. [ 3 ] for details). Therefore we determined the min-
imum required number of microbeads and the influence on the variance of the
ligand value.
Three microbead populations were loaded with different quantities of Atto
647N-labeled capture probes MLC-2v-cap or an artificial sequence designated as
aCS-cap, according to Rödiger et al. [ 3 ]. We found that the minimum number of
microbeads required for a reliable ligand value is about 20 per population, inde-
pendent of microbead population and capture probe. The reported signals were
uniform above 20 microbeads per population (Fig. 9 a, b). In this study we dem-
onstrated that about 20 microbeads are statistically sufficient for reliable test
results, a quantity not achievable by standard flow cytometry.
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