John M. S. Bartlett.pdf - Bio-Nica.info

132 Iannone et al.
6. Target concentration has a direct impact on signal intensity. Each panel in Fig. 2 includes
one negative control point. Additional points shown in the lower left are most likely the
result of PCR failure.
7. High salt concentrations help hybridization efficiency. However, we have found that
when analyzing samples on the LX-100, salt concentrations ≥400 mM result in optical
disturbances. This may be caused by the different refractive indices of the high ionic
strength core stream and the low ionic strength sheath fluid (14).
8. We have reserved one microsphere population and its associated cZipCode for use
with an SBCE positive control. Each SBCE reaction includes a short (40mer) synthetic
oligonucleotide target with a 4-fold degenerate position near its center and a complementary
capture probe to insure incorporation of reporter signal for any nucleotide assayed. Use
of this positive control in every SBCE assay well provides an excellent internal standard
to assess well-to-well reaction variability and can be used to normalize signal intensities
generated across a plate of 96 samples (14).
9. We have also used Rhodamine-6G (R6G)-labeled ddNTPs with the SBCE system on the
LX-100. Although using this directly coupled fluorochrome saves an additional wash and
incubation step, the fluorescent intensities were not as bright as PE. This is not unexpected
since the quantum efficiency of PE is much better than R6G.
10. Genotyping by OLA permits allele multiplexing and has no requirement for shrimp
alkaline phosphatase and Exonuclease I pretreatment of PCR target probes. The advantage
of SBCE is that only one capture probe is required for each allele, thus saving on costs.
The accuracy of genotyping using OLA or SBCE is >99% (7,8).
11. The inclusion of the detergent Tween 20 in the buffer helps reduce the loss of microspheres
during wash steps.
12. Because the LX-100 uses a dual-laser system for microsphere identification and reporter measurement,
spectral compensation is not required on the LX-100 as on the FACSCalibur.
13. Our current automated genotyping facility uses SBCE with 52 microsphere populations.
Enhanced throughput may be realized by incorporating multiplexed PCR amplifications.
14. Conversion of MFI to MESF, although not required, offers several advantages. These
advantages include (1) the use of a standard fluorescence unit; (2) the ability to compare
data between experiments and instruments; and (3) normalization of signal variability in an
instrument over time (caused by laser power shifts or PMT decline). Although not outlined
in this chapter, data from the LX-100 may also be converted to MESF using calibration
microspheres for PE. We have used QuantiBRITE PE beads from BD Biosciences for
this purpose.
15. Although this chapter focuses on the use of Luminex microspheres for multiplexed analysis,
there are other products available for developing multiplexed assays using a conventional
bench-top flow cytometer such as the FACSCalibur (see http://www.spherotech.com,
http://www.bangslabs.com). These products use one fluorescent parameter for the identification
of the microsphere populations and have a more limited level of multiplexing. For
manual acquisition of multiplexed assays on a conventional bench-top cytometer, (1) make
sure microspheres can be seen in forward versus side scatter and gate to exclude doublets;
(2) set the PMTs of fluorescent parameters to log acquisition; (3) adjust the PMT settings
of fluorescent parameters so that unstained and brightly stained microspheres are not offscale;
and (4) adjust compensation settings to subtract reporter fluorescence from the other
fluorescent channels. When properly compensated, microsphere populations with bright
reporter staining will not move out of the regions that identify their population.
16. Microsphere-based SNP genotyping by OLA or SBCE is an accurate and rapid method
for the analysis of SNPs.