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Troubleshooting Forum
Molecular Biology Techniques Q&A
This month’s questions from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) come from the “Microarrays” section. Entries have
been edited for concision and clarity. Mentions of specific products and manufacturers
have been retained from the original posts, but do not represent endorsements by, or the
opinions of, BioTechniques.
How can I eliminate false positive signals seen on my Luminex liquid microarrays? (Thread 15821)
While working on developing a Luminex liquid microarray-based assay, I encountered problems with microsphere cross-talk where certain microsphere regions on
my arrays are consistently misread. I use the Bio-Rad Bio-Plex machine and Luminex
FlexMAP beads. Is the problem likely caused by the hardware or should I focus on
the analyte? Has anyone experienced similar problems who can offer some troubleshooting ideas?
A I’m also developing a Luminex assay. What do you mean by microsphere cross-talk? What
value are you using to get your data? You should always use the median to remove any outlier
carryover from well-to-well. If you are using the median and still seeing poor results, you
might want to try to contact Bio-Rad technical support or have the machine serviced.
By cross-talk, I mean false-positive signals for regions other than the actual microsphere regions. For instance, bead 73 has been read as 22, or bead 44 has been read as 32.
It happens when a single analyte (bead type) is present in a reaction, but the machine is set
to analyze two or more analytes. Occasionally, the false-positive readings are thousands of
MFI, which is even higher than the real readings. It happens randomly and doesn’t occur
in all wells. I think that the optical system may not be aligned properly and is causing the
problem. If the problem was with the beads, they should be misread from wells. I don’t
think that carryover from well to–well is a possibility.
A Do you keep the beads out of the light? Could they be photobleached? I would get in
touch with the suppliers to discuss this. Bead carryover is only ~0.5% so using the median
would readily factor it out if that were your problem.
Beads 73 and 22 are at opposite ends of the CL1/CL2 scatter, so I don’t think that
this is cross-talk. I frequently see beads being read in the ‘next-up-right’ region (i.e., bead
22 read as bead 30). I have been able to eliminate this by gating appropriately. What DD
gate dimensions are you using? Why did you set the machine to look for more than one
region if you only have one region? If you only select the bead region that you are using
in your assay, it won’t matter if you get reads from other locations.
Carryover from well-to-well is a common problem, but I agree with the previous response
that reporting the median eliminates it. We have tested the extent to which well-to-well
carryover can occur and have seen beads carried over into 4 or 5 adjacent wells, although
carryover within 2 to 3 wells is most common. On one occasion, we saw a bead counted
16 wells away from the well containing that bead region.
Is the Luminex calibrated properly? If so, you don’t need to worry about optical alignment.
We even transported a machine in the back of a car and the lasers stayed aligned.
There are several reasons why beads appear in the wrong locations and easy solutions for
most of these problems:
1. Air in the system (protocols are available in the manufacturer’s information for
correcting this)
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2. Particles in the buffers (filter everything)
3. Damaged/bleached beads (check for
this by running surplus beads through
the Luminex)
4. Sample/matrix effects, but this is
Q I followed your recommendations and
my problem was indeed caused by carryover
from well-to-well. I had been analyzing
different samples with different analytes
in the same run. In order to do so, I set the
machine to read all those regions although
each sample included only a single analyte.
During analysis, I got readings from other
I was still confused by the magnitude of
fluorescent response from the contaminating
microspheres though. Although the number
of beads carried over was only in the tens or
singles, the MFI was as high as though it
was read from hundreds or thousands. The
default settings on the Bio-Rad Bio-Plex
software don’t show the number of beads
analyzed per well. When I looked into the
details of the raw data report, I saw that the
false-positive signals were coming from a
very few beads carried over from the other
reactions. The software seemed to somehow
approximate the fluorescence from those few
beads and extrapolate to the fluorescence
that would be seen from 100 beads, since
that is what I had as my reading setting. I
am looking into a way to change the settings
on the machine so that it won’t record a
fluorescent signal if the number of beads is
less than the limit set by the user. It should
just display error messages for low bead
numbers and reading problems instead of
extrapolating the fluorescence readings.
Q I am running a nucleic acid experiment.
I optimized the PCR and hybridized the
product before analyzing with the Luminex
machine. My no-template control and
background controls are reading as negative,
and the positive signals are reading in the
thousands. In my latest experiment, I ran
a dilution series of a plasmid control from
10⁹ through ten copies/reaction. I did not
see a difference in the level of fluorescence
from the highest control concentration
through the lowest. This surprised me
because I was under the impression that
Luminex technology was semi-quantitative.
I haven’t worked with nucleic acids in
Luminex assays before, but I will see if I can
be of any help based on my other Luminex
experience. With protein coating, you can
get semi-quantitative results, so I would
assume the same is true with nucleic acids.
When you say you did a plasmid dilution
series, was this a PCR template dilution?
Even with lower concentrations, you may
still be producing enough product to saturate
your beads. Try diluting your product to see
if you can get a quantitative result. If your
beads are a commercial product, you might
try contacting the supplier.
A When you analyze the tubes after the
PCR, they will all tend to have reached a
maximum. By that, I mean that each sample
has a high and fairly uniform amount of
DNA amplicon. When you analyze these
products, you will find this uniform high
level, independent of the method of analysis.
Think of gels, for example. When you run a
gel after a 30- or 35-cycle PCR, you see bands
that are about the same intensity or you see an
absence of bands in the negative controls.
The Luminex instrument is quantitative
from zero MFI up to about 25,000 MFI. If
you want your PCR to be quantitative, you
must use a much lower cycle number than
usual. This is not necessarily related to the
detection method (gel, Luminex, or other),
but is a function of the exponential accumulation of amplicon in the tube during PCR.
It might help if you see some Luminex
standard curves. These are readily available
if you search Google Images.
Selected and edited by Kristie Nybo, Ph.D.
BioTechniques 49:703-705 (October 2010)
doi 102144/000113511
Why don’t the fluorescence intensities
decrease with reduced concentration
of PCR product? (Thread 17191)
I am running a sensitivity study with
the Luminex system. I get a fluorescent signal
from 10⁹ through 10 copies/reaction, but I do
not see a significant decrease in signal from
the highest concentration to the lowest.
What causes this?
What does your negative control look
like? Have you optimized the antibody
concentration? If you provide more details
on what troubleshooting steps you have tried
already, I might be able to help since I often
work with Luminex arrays.
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