3DNA Array 900, Array 350 and Array 50 (Version 2) Kit Troubleshooting Guide   Section III. Background Problems   Symptom Cause Resolution 1. High, uniform background (in excess of 200-300 counts, one or both channels). A. Array is aged or improperly stored. As arrays age, some surface chemistries, particularly poly-L-lysine, show an increase in auto-fluorescence in the Cy3 channel. Aged arrays can show a higher affinity for salt which will fluoresce in the Cy3 channel. Proper storage of the arrays (vacuum dessicated) is recommended. Scan array prior to hybridization to determine basal level of background on the array. Note age of arrays prior to use. For further recommendations for reducing background, refer to the Appendices of the protocols.   B. Coating chemistry. Some array surface chemistries, including poly-L-lysine, aldehyde, aminosilane, epoxy or mirrored slides, may be prone to a high, uniform background in either channel. Slide prehybridization (blocking) and increasing the wash times may reduce the background observed. In addition, a particular combination of surface chemistry and hybridization buffer may yield lower background than others. Scan array prior to hybridization to determine basal level of background on the array. Please consult with Genisphere Technical Support for a list of buffer components that may be incompatible with the 3DNA reagent. For further recommendations for reducing background, refer to the Appendices of the protocols.   C. Use of poor quality DTT in washes. Use of poor quality DTT in wash buffer (often used to prevent Cy5 oxidation) can contribute to high, green background. Use fresh DTT for treating wash buffers.   Symptom Cause Resolution 2. High, non-uniform background (one or both channels, portions of the array, can appear as fluid-like or swirls). A. Uneven slide coating. Tends to occur in printing batches and is not normally resolvable. Always use, in any one experiment, successive arrays from the same printing batch. Scan array prior to hybridization to determine basal level of background on the array. B. Array was not appropriately dried after a wash series. After any wash series (post hybridization or blocking procedure), QUICKLY transfer the microarray to a 50 ml conical tube and centrifuge at 1000 rpm for 2-3 minutes. Any buffer left on the array will dry resulting in high, green background. Rewashing the array may help to eliminate this type of background. C. Residual SDS or salt on array surface. SDS will fluoresce in the Cy5 channel while salt will fluoresce in the Cy3 channel. Use high quality SDS and SSC for preparation of wash buffers and filter sterilize. Avoid any delay in the final spin step after the washes. Extending the wash times may help to prevent SDS or salt from remaining on the array. D. Bar code, ink or marker was used to label the array. Avoid labeling array with tags, pens or markers which may cause background fluorescence. For example, a blue pen or marker will result in high Cy5 (red) channel background. Use a glass etching pen to label the array. If bar code is used, spin dry with bar code LABEL DOWN in conical tube.   E. Unlevel hybridization. Uneven background can result from hybridizations that are not completely horizontal. Make certain that the array surface is level (front to back and side to side).   F. Air bubbles under cover slip. Air bubbles can form under coverslips which can lead to high background and low specific signal. Avoid air bubbles when placing coverslip on array. See section II.5 for further details on avoiding air bubbles.   G. Bad Hybridization Buffer. Hybridization Buffers provided by Genisphere contain components that are prone to degradation if improperly handled or stored (or are beyond the expiration date). Use of such buffers in microarray experiments may result in high background. For example, Denhardt's solution, when degraded, can show an increase in red channel background. Avoid use of questionable hybridization buffers.   Symptom Cause Resolution 3. Grainy Background. A. Array dried during hybridization. Make certain the hybridization chamber is adequately humidified, sealed and the appropriate volume is used under the coverslip. If using Enhanced Hybridization Buffer (for cDNA hybridization only), increasing the volume by 10 to 15% may be necessary to adjust for volume loss in pipet tip. If the SDS based hybridization buffer (Vial 6) or Enhanced Hybridization Buffer was used, try using the formamide-based buffer (vial 7) at a lower temperature. For further details, see the “cDNA Hybridization” and "3DNA Hybridization" sections of the protocol and Appendix C of the Troubleshooting Guide for recommended hybridization volumes for various size coverslips. B. Coating chemistry. Some array surface chemistries, including poly-L-lysine, aldehyde, aminosilane, epoxy or mirrored slides, may be prone to a high, uniform background in either channel. Slide prehybridization (blocking) and increasing the wash times may reduce the background observed. In addition, a particular combination of surface chemistry and hybridization buffer may yield lower background than others. Scan array prior to hybridization to determine basal level of background on the array. Please consult with Genisphere Technical Support for a list of buffer components that may be incompatible with the 3DNA reagent. For further recommendations for reducing background, refer to the Appendices of the protocols. C. Array is aged or improperly stored. As arrays age, some surface chemistries, particularly poly-L-lysine, show an increase in auto-fluorescence in the Cy3 channel. Aged arrays can show a higher affinity for salt which will fluoresce in the Cy3 channel. Proper storage of the arrays (vacuum dessicated) is recommended. Scan array prior to hybridization to determine basal level of background on the array. Note age of arrays prior to use. For further recommendations for reducing background, refer to the Appendices of the protocols. D. Wash buffer impurities or inadequate washing. Wash buffer impurities can cause grainy background. Use of high quality reagents and filtering the buffers will help to eliminate this problem. Insufficient washing may also contribute to grainy background. Increasing the wash times by 5 minutes and/or slight agitation may help to improve the background.   E. Capture reagent not fully resuspended. Under normal storage conditions, the dendrimer molecules can form aggregates that may stick to the array surface. Follow the resuspension protocol thoroughly. For further details, refer to the "3DNA Hybridization" section of the protocols.   F. Hybridization buffer solution not fully resuspended. Make certain buffers are thoroughly resuspended. After resuspension, spin buffers briefly to collect particulates at the bottom of the tube. Avoid decanting from the bottom. For further details, refer to the "cDNA Hybridization " section of the protocols.   G. Scanner laser and/or PMT settings too high. All array surfaces have a certain level of auto-fluorescence. Increasing the scanner laser and/or PMT settings to adjust for low specific signal will cause an increase in background. To generate higher specific signal, increase the amount of RNA used in the cDNA synthesis reaction. Avoid setting the scanner too high. For a further explanation of signal and background noise, consult the scanner manufacturer. Refer to Low/Absent Signal section of the Troubleshooting Guide.   Symptom Cause Resolution 4. Array coating damage (peeling, cracking, or missing portions of the surface coating). A. Surface coating susceptibility. Some array surfaces, particularly poly-L-Lysine, tend to become unstable over time (consult array provider for recommended storage conditions). The process of hybridization and washing can cause these surfaces to crack and peel off of the glass surface, observed as extremely clean (nonfluorescent) areas on the array. Avoid using aged arrays. In cases where aged arrays must be used, vial 7 hybridization buffer at the lower temperature is recommended. Avoiding the EtOH wash when using aged and poly-L-lysine coated arrays may help to reduce damage to the coating. For further details, refer to the "cDNA Hybridization" and "3DNA Hybridization" sections of the protocols. If problem persists, contact Technical Support for additional buffer recommendations. B. Physical contact of object with array surface. Take extreme care when handling the array. Avoid surface contact with objects such as pipet tips. Damage to the array surface often occurs while floating the coverslip off of the array in solution. Allow coverslip to float off of the surface. DO NOT attempt to pry off the coverslip as this will likely damage the array. Use appropriate volume under coverslip and properly humidify and seal the hybridization chamber to avoid drying. C. Drying of hybridization solution under cover slip. Drying under the coverslip often results in damage to the array surface upon removal of the coverslip. Any difficulty in removing the coverslip from the array indicates drying. Use appropriate volume under coverslip and properly humidify and seal the hybridization chamber to avoid drying. Soaking the array for several minutes, with slight agitation, in the first wash buffer at either 42oC (oligo arrays) or 65oC (cDNA arrays) can help in the removal of a stuck coverslip.   D. Excessive amount of SDS in hybridization. Too much SDS can damage some array surfaces. If determined to be the cause of array damage, use of an alternative hybridization buffer with lower SDS content, such as vial 7, is recommended.   Symptom Cause Resolution 5. Visible haze on array surface. A. Precipitation of salt onto array during EtOH wash. (Array 350 and Array 50 V2 kits). Salt from the final wash buffer (0.2X SSC), after cDNA hybridization, precipitates out of solution onto array surface upon submersion into 95% EtOH. Likely a result of a higher than intended concentration of salt in 0.2X SSC buffer. Accidentally switching the second and third washes can cause this phenomenon. A brief rewash in 0.2X SSC followed by EtOH wash will likely remove salt from the array. If the salt is not removed, repeat 0.2x SSC wash, skipping EtOH wash. Avoid the EtOH wash for future experiments (current protocols reflect elimination of this step). B. Array was not appropriately dried after a wash series. After any wash series (post hybridization or blocking procedure), QUICKLY transfer the microarray to a 50 ml conical tube and centrifuge at 1000 rpm for 2-3 minutes. Any buffer left on the array will appear as haze on array and result in high, green background.

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