|
Symptom |
Cause |
Resolution |
|
BACKGROUND / HETEROGENEITY |
|
High Background |
Hybridization system |
If you are using a hybridization system, please make sure you are following the manufacturer’s
instructions carefully. Incorrect use of these products can lead to a variety
of problems (see heterogeneity below). |
| |
Degraded reagents |
It is always important to utilize high-quality reagents when performing hybridization
experiments. In particular, a fresh stock of deionized formamide is essential
to effective hybridization. Note that, unlike other products, formamide is
not included in the OneArray pre-hybridization buffer. |
| |
Improper protocol |
Phalanx Biotech scientists have modified the pre-hybridization protocol designed to
improve signal-to-noise ratio. This protocol is available in the latest
version of the User Guide at the Phalanx Biotech website: http://www.phalanxbiotech.com/Support/Support.html. |
| |
Improper pre-hybridization buffer |
It is essential to use the proper amount of BSA during pre-hybridization. The
BSA should be at least molecular biology grade, as electrophoresis grade
leads to insufficient blocking. In addition, the concentration must be 1%
BSA, or 10 mg/mL. |
Graininess
(click to enlarge)
|
Column impurities |
Purification
of labeled aRNA on elution columns can sometimes generate detectable
impurities in the sample. This problem can be alleviated by purifying larger
batches of aRNA on a column. Then, when smaller amounts of aRNA are used for
hybridization, the impurities are diluted, eliminating the grainy
interference on the microarray. |
| |
Unfiltered solutions |
Solutions
must be free of impurities. This is especially important for the wash
solutions, as trace impurities can adhere to the slide. The best way to avoid
this problem is to filter all wash solutions prior to use. |
Slide
Heterogeneity
(click to enlarge)
|
Hybridization system |
The most likely cause of this type of heterogeneity is improper hybridization. If
the target sample is not allowed to interact fluidly with the entire array
surface, uneven signal will result. Some hybridization systems are
susceptible to this type of problem when not used properly; please read the
manufacturer’s instructions carefully. |
(click to enlarge)
|
"Black Holes" |
The
most likely cause of this type of heterogeneity is the presence of an air pocket under the coverslip. These are often easy to recognize as a circular region that shows lack of hybridization. Take care that no bubbles are present to ensure an even hybridization. |
(click to enlarge)
|
Improper Washing |
The
most likely cause of this type of heterogeneity is improper washing of the microarray
slide. This may occur during pre-hybridization, hybridization, or
post-hybridization:
- The most likely cause of this problem is improper post-hybridization
washing. Be sure the mixtures are in correct proportion and fresh reagents
are used. Temperatures and wash times are essential to proper
post-hybridization; if performing multiple hybridizations simultaneously,
take care that all microarrays are washed thoroughly. Finally, when working
with multiple arrays, keep the arrays in a solution of room temperature 2X
SSPE to avoid drying. We recommend having a secondary container (staining
dish) for this purpose.
- During pre-hybridization, residual dye may be deposited on the surface of
the microarray if the slide is not washed and dried carefully. A pre-scan of
the slide immediately following pre-hybridization may reveal this is the
problem. The signal will likely be much weaker than that seen the example
above. If this is the case, make sure the solutions are fresh and in the
correct proportion, that all temperatures are correct, and that the timeline
for rinsing and drying is followed properly.
|
| |
Array drying |
During
hybridization, the microarray surface must remain saturated with hybridization
buffer at all times. If using the Phalanx Biotech protocol, ensure the
hybridization chamber has sufficient SSPE buffer to keep the local atmosphere
saturated. If using a hybridization system, carefully review and follow the
manufacturer’s instructions. |
Spot
Heterogeneity
(click to enlarge)
|
Mishandling |
Phalanx Biotech scientists have modified the pre-hybridization protocol designed to
improve signal-to-noise ratio. This protocol has also been found to help in
resolving many spot morphology issues. The protocol is available in the
latest version of the User Guide at the Phalanx Biotech website: http://www.phalanxbiotech.com/Support/Support.html.
Another source of this shape can be mishandling of the microarrays. This
applies to all handling, but may be particularly relevant if using the Round
Cap Tube supplied with your OneArray microarray. When handling the slides, be
careful not to drop them into place; slide them carefully and gently.
Excessive jarring can cause changes in spot morphology that may lead to
compromised data quality.
|
Poor
signal
(click to enlarge)
|
Slide scanned incorrectly |
Take
care to load the slide into the scanner with the proper orientation. |
|
DATA ISSUES |
|
Poor differential signal |
Non-specific binding |
Non-specific binding can be the result of
problems at various stages of the process:
- Hybridization: ensure the temperature for hybridization is correct; an
excessively low temperature can lead to non-specific binding.
- Washing: again, a common cause is an improper temperature. Ensure the
initial wash is performed using 2X SSPE / 0.1% SDS at 42°C.
|
| |
High PMT setting |
Check
the settings for your scanner to ensure the PMT is not too high, as this can
lead to a significant increase in signal leading to saturation. |
|
SIMPLE MEASURE PROGRAM |
|
SimpleMeasure does not start |
Java not installed |
Ensure you have an up-to-date version of
Java on your computer. This can be found on the Phalanx Biotech website at http://www.phalanxbiotech.com/Support/SimpleMeasure_dwnloads.html. |
|
SimpleMeasure does not finish processing |
Improper file format |
Simple measure cannot read your input
file. The only file types allowed are GenePix *.gpr files. Furthermore, the
program will only process dual-color scans, so be sure you are scanning both
the Cy3 and Cy5 wavelengths and that these are both included in the saved
*.gpr file to be analyzed. |
