Troubleshooting Guide

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 Support Page.
	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 Support Page. 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 here.
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.