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3DNA Array 900MPX and Array 350RP Kits Troubleshooting Guide |
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Section IV. Signal Problems
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| Symptom |
Cause |
Resolution |
| 1. Low/absent signal in both channels. |
A. Array was improperly spotted or processed. |
High quality microarray results are achievable only when the target DNA molecules are properly spotted and available for hybridization! Confirm that the spotted nucleic acids are present and available for hybridization by using one of the published techniques for generically detecting nucleic acid on the array. For example, use Genisphere’s Universal Feature Detection Kit to confirm the quality of arrays prepared from PCR-amplified cDNAs. Use of common processing protocols is recommended. Avoid treatment of arrays with succinic anhydride as this will interfere with Genisphere labeling. For further information, consult Genisphere's Technical Support. |
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B. Failed/poor cDNA synthesis: bad reverse transcription component. |
All of the components in the reverse transcription reaction (RT enzyme, dNTPs, reaction buffer, and DTT) are vital to efficient cDNA synthesis. Failure of any one of these components will result in a failed synthesis reaction. Properly store and handle all components. DO NOT use expired reagents. To check the efficiency of cDNA synthesis, run an aliquot (1 ug of RNA equivilent) on an acrylamide gel (6-10%), stain with SybrGold (Molecular Probes). See Appendix B of the Troubleshooting Guide for details on running cDNA gels. |
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C. Failed/poor cDNA synthesis: incompatible RT enzyme. |
Genisphere has tested several of the many reverse transcriptase enzymes available on the market. Our studies have shown that these enzymes have different attributes and variable performance characteristics, particularly among various species. Check peer-reviewed articles to determine which enzyme is commonly used for a species of interest. If this information is not available, Superscript II (Life Technologies), a widely used RT enzyme for microarrays, is recommended. |
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D. Failed/poor cDNA synthesis: RNA sample contains RT inhibitor, genomic DNA, or oligo dT. |
Because all RNA samples are unique and RNA purification methods vary, inhibitors of reverse transcriptase (polysaccharides, polyphenols, etc.) may co-purify with total RNA samples, resulting in poor cDNA synthesis. It may be necessary to further purify the RNA sample by ethanol precipitation. Genomic DNA can interfere with reverse transcription by competing with RNA for enzyme binding sites. Genomic DNA should be removed from sample by using a ribonuclease-free DNase. MAKE CERTAIN DNASE IS FULLY INACTIVATED PRIOR TO CDNA SYNTHESIS! Lastly, RNA samples that have been purified with dT beads or columns (poly A+ RNA) may contain varying amounts of residual dT that may compete with Genisphere primers resulting in weak signal (Leeb et al., BioTechniques 34:720-724, April 2003). In this case, use of the higher concentration primer (included in the kits) is recommended. For further details, refer to the “RNA Preparation” and Appendices sections of the protocols. |
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E. Failed/poor TdT reaction: bad TdT component (Array 900 MPX kit).
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Failure of the TdT reaction (either completely or partially) will prohibit subsequent ligation of the capture sequence to the cDNA. The components of the TdT reaction (TdT enzyme and dTTP) are volatile. Failure of either of these components will result in weak or no signal in one or both channels. DO NOT use expired reagents. Replace reagents if suspect. |
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F. Failed/poor ligation reaction: bad ligation component.
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Attachment of the "capture sequence" to cDNA is necessary for signal generation. The components of the ligation reaction (ligase and ligation mix) are volatile. Failure of either of these components will result in weak or no signal in one or both channels. DO NOT use expired reagents. Replace reagents if suspect. |
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G. Inefficient/failed purification step (after either cDNA synthesis or ligation reaction). |
Failure of the Qiagen purification columns will result in little or no signal in one or both channels. Refer to section II, Procedural Problems, of the Troubleshooting Guide, for more information about purification column failures. |
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H. Inefficient/failed concentration of cDNA (Array 350RP kit). |
Two methods are described in the protocol for concentration of cDNA post reverse transcription: EtOH precipitation and Microcon concentration. Of the two methods, Genisphere highly recommends the use of Microcon columns for reproducibility and efficiency. However, all methods of concentration can have an occasional failure. If using the Microcon method, be certain to prewet the column, as described in the protocol. If concentrating by EtOH precipitation, follow the protocol closely. Aspirate the supernatant with a pipette rather than decanting. If the pellet has not completely adhered to the side of the tube, it can easily be lost. Avoid aspirating small pieces of pellet. Once the pellet is dried, resuspend by heating to 65oC and vigorously mixing. Make certain to use a co-precipitant, preferably linear acrylamide (included in kits) or glycogen as opposed to tRNA (may interfere with hybridization). Non-fluorescent pellet paint (Novagen) may be used as a co-precipitant to aid in visualization of pellet. For further details, refer to the “Ethanol Precipitation” and “Hybridization “ sections of the protocols (Array 350 and 50 V2 kits). |
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I. RNA sample is contaminated with ribonuclease, proteins, or genomic DNA. |
The integrity of all RNA samples should be confirmed by gel electrophoresis (agarose) prior to use (see Appendix A of the Troubleshooting Guide for a protocol for running RNA gels). Multiple freeze/thaw cycles can degrade RNA. RNase inhibitor or RNA Later (Ambion) should be added to stored RNA samples suspected of being contaminated with RNase. Inhibitor should also be added during the reverse transcriptase reaction to avoid RNA degradation during cDNA synthesis (added after primer annealing). Other contaminants, such as proteins and genomic DNA, can cause miscalculations of RNA quantity (particularly when relying on absorbance spectroscopy). Running a gel with the appropriate controls is recommended for determining purity and concentration. For further details refer to the “RNA Preparation” section of the protocol. |
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J. Capture Reagent (vial 1) was not used or used in insufficient amounts.
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Since the cDNA is not labeled during the RT reaction, it is necessary to include the fluorescent labeled Capture Reagent, Vial 1, in the 3DNA hybridization step; otherwise, no signal will be visible on the array. Repeat the 3DNA hybridization procedure using 2.5 ul of the Capture Reagent. Make certain that the Capture Reagent is thoroughly resuspended by heating and vortexing. For more information, refer to the "3DNA Hybridization" section of the protocol. |
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K. Genisphere hybridization buffers not used. |
Use of hybridization buffers other than those provided in the kit may compromise results, adversely affecting the 3DNA hybridization. Dextran Sulfate, used in excess of 2%, causes the dendrimer arms to precipitate out of solution, resulting in little or no signal. Salt concentration should fall within the range of 250-500 mM final (or between 2-4X SSC). If, for some reason, the provided buffers cannot be used, check with Technical Support for alternative buffer recommendations. |
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L. Hybridization or wash temperature too high (cDNA or 3DNA hybridizations). |
The hybridization and wash temperatures are critical for a successful result in any array experiment. Excessive temperatures may cause low signal intensity and even cause some features to come off the array. Confirm the hybridization temperature of the incubator or water bath with a calibrated thermometer. Conversely, inappropriately low hybridization and wash temperatures will yield poor differentials by promoting nonspecific binding. The temperatures outlined in the protocol are intended to be a starting point for most users. For best results, the optimal temperatures should be determined by the end user. For more information, refer to the "cDNA Hybridization" and "3DNA Hybridization" sections of the protocols. |
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M. Hybridization too short (cDNA or 3DNA hybridizations). |
The hybridization times for both of the cDNA and 3DNA hybs have been closely studied and optimized. However, the completion of the reactions will depend on factors such as temperature and buffer composition. Increasing the hybridization times may help to increase signal. Please consult Tech Support for more information on this subject. |
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N. Array dried during hybridization. |
Make certain the hybridization chamber is adequately humidified, sealed and the appropriate volume is used under the coverslip (see Appendix C of the Troubleshooting Guide for recommended hybridization volumes for various size coverslips). 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 protocols. |
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O. Excessive competitor DNA used. |
Condensation of humidifying solution onto array surface in hybridization chamber can severely reduce signal. Prewarm array, hyb solution and hyb chamber to prevent this phenomenon. |
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P. Array was hybridized on wrong side. |
Ensure the correct side of the array is hybridized. |
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Q. Condensation on array surface during hybridization. |
Condensation of humidifying solution onto array surface in hybridization chamber can severely reduce signal. Prewarm array, hyb solution and hyb chamber to prevent this phenomenon. |
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R. Scanner problems. |
The proper functioning of the scanner hardware should be determined prior to use. Check the scanner focal point and determine that the lasers are functioning appropriately for all channels. Many manufacturers provide a calibration slide with the instrument. Make certain that the slide has been inserted correctly. For more information, consult the scanner manufacturer. |
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| Symptom |
Cause |
Resolution |
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| 2. Low/absent signal in one channel. |
A. One RNA sample is contaminated with ribonucleases, proteins, or genomic DNA.
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The integrity of all RNA samples should be confirmed by gel electrophoresis (agarose) prior to use (see Appendix A of the Troubleshooting Guide for a protocol for running RNA gels). Multiple freeze/thaw cycles can degrade RNA. RNase inhibitor or RNA Later (Ambion) should be added to stored RNA samples suspected of being contaminated with RNase. Inhibitor should also be added during the reverse transcriptase reaction. Other contaminants, such as proteins and genomic DNA, can cause miscalculations of RNA (particularly when relying on absorbance spectroscopy). In addition to absorbance spectroscopy, gel electrophoresis is recommended for determining purity and concentration of the RNA sample. For further details refer to the “RNA Preparation” section of the protocol. |
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B. Failed/poor cDNA synthesis: RNA sample contains RT inhibitor, genomic DNA, or oligo dT.
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Because all RNA samples are unique and RNA purification methods vary, inhibitors of reverse transcriptase (polysaccharides, polyphenols, etc.) may co-purify with total RNA samples, resulting in poor cDNA synthesis. It may be necessary to further purify the RNA sample by ethanol precipitation. Genomic DNA can interfere with reverse transcription by competing with RNA for enzyme binding sites. Genomic DNA should be removed from sample by using a ribonuclease-free DNase. MAKE CERTAIN DNASE IS FULLY INACTIVATED PRIOR TO CDNA SYNTHESIS! Lastly, RNA samples that have been purified with dT beads or columns (poly A+ RNA) may contain varying amounts of residual dT that may compete with Genisphere primers resulting in weak signal (Leeb et al., BioTechniques 34:720-724, April 2003). In this case, use of the higher concentration primer (included in the kits) is recommended. For further details, refer to the “RNA Preparation” and Appendices sections of the protocols. |
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C. Failed/poor TdT reaction: bad TdT component (Array 900 MPX kit).
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Failure of the TdT reaction (either completely or partially) will prohibit subsequent ligation of the capture sequence to the cDNA. The components of the TdT reaction (TdT enzyme and dTTP) are volatile. Failure of either of these components will results in weak or no signal in one or both channels. DO NOT use expired reagents. Replace reagents if suspect. |
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D. Failed/poor ligation reaction: bad ligation component.
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Attachment of the "capture sequence" to cDNA is necessary for signal generation. The components of the ligation reaction (ligase and ligation mix) are volatile. Failure of either of these components will results in weak or no signal in one or both channels. DO NOT use expired reagents. Replace reagents if suspect. |
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E. Only one Ligation Mix used for dual color experiment. |
Each Capture Reagent, the reagent containing the dyes, requires a specific capture sequence attached to the cDNA to provide signal to the array. For example, for a sample to be labeled with the Cy3 Capture Reagent, it must contain the Cy3 capture sequence (attached during the ligation reaction) and likewise for labeling a sample with Cy5. Make certain to use the correct Ligation Mix to label each sample. |
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F. Capture Reagent (vial 1) was not used or used in insufficient amounts.
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Since the cDNA is not labeled during the RT reaction, it is necessary to include the fluorescent labeled Capture Reagent, Vial 1, in the 3DNA hybridization step; otherwise, no signal will be visible on the array. Repeat the 3DNA hybridization procedure using 2.5 ul of the Capture Reagent. Make certain that the Capture Reagent is thoroughly resuspended by heating and vortexing. For more information, refer to the "3DNA Hybridization" section of the protocols. |
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G. Scanner problems.
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The proper functioning of the scanner hardware should be determined prior to use. Check the scanner focal point and determine that the lasers are functioning appropriately for all channels. Many manufacturers provide a calibration slide with the instrument. Make certain that the slide has been inserted correctly. |
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H. Plastic coverslip was used. |
Cy5 binds to plastic. Therefore, when plastic coverslips are used, some or all of the Cy5 labeled 3DNA reagents and cDNA will bind to the coverslip, removing them from the hybridization. Use a glass coverslip. |
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I. Cy5/Alexa Flour 647 oxidation. |
Cy5 and Alexa Flour 647 fluors tend to oxidize and degrade more rapidly than Cy3. To prevent oxidation, add anti-fade reagent (Vial 8) to the hybridization buffer, as described in the protocol. In addition, use RNase-free deionized distilled water for wash buffer preparation. Deionized (DI) water produced from canister type systems can oxidize Cy5 and Alexa Flour 647. Finally, add DTT to the wash buffer (0.1mM – 1.0 mM). Lastly, if dye damage is determined to be due to exposure of atmospheric pollutants (ex. ozone), use Genisphere's DyeSaver product. For additional information regarding stabilizing Cy5 and Alexa Flour 647, refer to the Appendices of the protocols or contact Technical Support. |
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J. Fluor photo-bleaching. |
Many fluors are susceptible to degradation through repeated excitation/emission cycles, known as photo-bleaching. Repetitive scans and excessive laser voltage settings may cause photo-bleaching. Reducing the number of scans and scanner settings will prolong the integrity of the fluors. Use of Genisphere's DyeSaver product has also been shown to significantly reduce laser induced photo-bleaching. Please contact Technical Support for further information on this topic. |
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| Symptom |
Cause |
Resolution |
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| 3. Uneven signal across array. |
A. Array dried during hybridization. |
Make certain the hybridization chamber is adequately humidified, sealed and the appropriate volume is used under the coverslip (see Appendix C of the Troubleshooting Guide for recommended hybridization volumes for various size coverslips). 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 protocols. |
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B. Condensation on array surface during hybridization. |
Condensation of humidifying solution onto array surface in hybridization chamber can severely reduce signal. Prewarm array, hybridization solution and hybridization chamber to prevent this phenomenon. |
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C. Unlevel hybridization. |
Uneven signal can result from hybridizations that are not completely horizontal. Make certain that the array surface is level (front to back and side to side). |
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D. RNA sample contains contaminants that inhibit uniform hybridization. |
Phenols, polysaccharides and other contaminants within the RNA sample can cause nonuniform hybridizations if not removed. A purification column, such as Qiagen's QIAquick PCR purification column, after cDNA synthesis, may be needed. |
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E. cDNA not fully resuspended (Array 50V2 and 350 kits). |
The Array 350RP kit requires concentrating the cDNA prior to hybridization to the array. Inadequate resuspention of cDNA in the hybridization mixture can result in uneven signal. Make certain that the cDNA is thoroughly resuspended prior to hybridization. |
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