3DNA Array 900MPX and Array 350RP Kits Troubleshooting Guide   Section II. Procedural Problems   Symptom Cause Resolution 1. Unable to carry out reverse transcription reaction in recommended volume. A. RNA concentration is too dilute. The minimum concentration of total RNA required by each of the kits, as outlined in the kit protocols, is as follows: 0.4 ug/ul (Array 900MPX) and 0.2 ug/ul (Array 350RP). These values are based on the highest recommended input amounts for each of the kits (ex. 4ug for the Array 900MPX kit). Lower concentrations may be used if using lower input quantitities. If using RNA that is too dilute to achieve the outlined reaction volume, concentrate the sample by a suitable method (ex. NH4OAc/EtOH precipitation).   B. Attempting to use more RNA than is called for in the protocol. Some arrays may require more input RNA than is called for in the protocol. If the desired quanitity of input RNA exceeds the volumes outlined in the protocols, the enzymatic reactions can be scaled linearly. Note: scaling up may require purchase of additional reagents. DO NOT exceed load limitations of the Qiagen columns (10 ug of DNA for the PCR Purification column and 5 ug of DNA for the MinElute Purification column). Symptom Cause Resolution 2. Excessive elution volume (Qiagen clean-up columns). A. Used Qiagen PCR Purification Column instead of Qiagen MinElute Column (Array 900MPX kit). Array 900MPX calls for use of Qiagen's MinElute columns (elution volume of 10ul) for clean-up of reverse transcription and ligation reactions. If using the PCR Purification Column (elution volume of 50ul), the elution volume will need to be reduced (ex. drying) or the subsequent reaction volumes will need to be scaled-up accordingly (will also require concentration of samples prior to hybridization). Call Tech Support for more information. B. Failed Qiagen Column. May result from bad buffers or column. Repeat assay. Be certain to store all reagents according to the manufacture's recommendations. Symptom Cause Resolution 3. Low elution volume (Qiagen clean-up columns). A. Used Qiagen MinElute Column instead of Qiagen PCR Purification Column (Array 350RP kit). Although Array 350RP kit calls for use of Qiagen's PCR Purification column (elution volume of 50ul) for clean-up of reverse transcription and ligation reactions, the MinElute column (Qiagen) can be substituted (elution volume of 10 ul). In fact, use of the MinElute column for clean-up of the ligation reaction may eliminate the need of a concentration step. If using Qiagen's MinElute column to clean-up the reverse transcription reaction, adjust elution volume with water accordingly. Call Tech Support for more information. B. Failed Qiagen Column. May result from bad buffers or column. Repeat assay. Be certain to store all reagents according to the manufacture's recommendations. Symptom Cause Resolution 4. Microcon column: low volume or no volume recovered (Array 350RP kit). A. Failure to pre-wash the column. See step 2 of the "Concentration of cDNA with Millipore Microcon…" section of the protocol. Repeat assay. B. Failure to cap the column prior to the concentration spin. Will cause the column to dry, preventing recovery of sample. See step 6 of the "Concentration of cDNA with Millipore Microcon…" section of the protocol. Repeat assay. C. Failure to add 5ul of 1X TE to reservoir membrane and gently mixing prior to the elution spin. See step 9 of the "Concentration of cDNA with Millipore Microcon…" section of the protocol. Repeat assay. D. Failure to invert the column during the elution spin. See step 10 of the "Concentration of cDNA with Millipore Microcon…" section of the protocol. Invert column and respin. Symptom Cause Resolution 5. Microcon column: reservoir collapsed into collection tube during elution spin (Array 350RP kit). A. Excessive elution spin time. Repeat assay, reducing the elution spin time accordingly. Avoid spinning less than 30 seconds as this may result in incomplete recovery of sample. See step 10 of the "Concentration of cDNA with Millipore Microcon…" section of the protocol. B. Failed Microcon column. Repeat assay. Symptom Cause Resolution 6. EtOH precipitation: no visible pellet (Array 350RP kit). A. Lack of cDNA. Precipitating nanogram quanitities of DNA can be difficult and requires good technique for reproducibility. Pellets may disperse across one side of the tube and appear as a haze. Use of a coprecipitant such as linear acrylamide is useful in improving precipitation efficiency as well as visibility of the pellet. Run an aliquot of the cDNA (prior to concentration) on a gel to confirm successful cDNA synthesis (see Appendix B of the Troubleshooting Gude). If gel reveals low or no cDNA, repeat cDNA synthesis. B. Failed precipitation procedure. Precipitating nanogram quanitities of DNA can be difficult and requires good technique for reproducibility. Pellets may disperse across one side of the tube and appear as a haze. Use of a coprecipitant such as linear acrylamide is useful in improving precipitation efficiency as well as visibility of the pellet. Run an aliquot of the cDNA on a gel, both before and after concentration, to confirm a successful concentration step (see Appendix B of the Troubleshooting Guide). If gels confirm a failed precipitation procedure, make up fresh precipitation reagents (salt solution and EtOH) and repeat cDNA synthesis and concentration steps.     Symptom Cause Resolution 7. Formation of bubbles under coverslip. A. Drawing air into pipet tip. Set pipet volume to avoid drawing air into tip when applying hyb solution to array. B. Poor array loading technique. Good loading technique is essential to preventing bubble formation. We recommend applying hybridization solution across the surface of the array dropwise, placing one edge of the coverslip on the array and slowly lowering the other edge until the coverslip is flush. Practice this procedure with blank slides and hyb buffer to perfect the technique. Alternatively, if using Lifter Slips (Erie Scientific), the use of capillary action may help to prevent bubble formation. See Appendix C of the Troubleshooting Guide for tips on loading arrays. C. Hybridization solution and/or array not adequately pre-warmed. Due to the viscous nature of the hybridization buffers, pre-warming the mixture as well as the array prior to hybridization will allow the hybridization mix to spread evenly and help prevent bubble formation. D. Debris on coverslip or array surface. The presence of debris (glass fragments, dust etc.) may create bubbles by preventing the coverslip from sitting flush against the array surface. Make certain that both the array and coverslips are free of debris prior to applying hybridization mixture.     Symptom Cause Resolution 8. Coverslip stuck to array surface (either cDNA or 3DNA hybridizations). A. Drying of hybridization mixture: chamber not properly sealed or humidified. Make certain that the hybridization chamber is properly sealed and humidified during hybridization incubation. B. Inadequate volume loaded for size coverslip. See Appendix C of the Troubleshooting Guide for recommended hybridization volumes for various size coverslips. C. Use of Enhanced Hybridization Buffer. Enhanced Hybridization Buffer has been designed to include proprietary size-exclusion components. As a result, this buffer is significantly more viscous than the SDS and Formamide based buffers. Due to its high viscosity, Enhanced Hybridization Buffer is prone to a greater loss of volume in the pipet tip. This in turn can promote sticking of the coverslip to the array during the cDNA hybridization. 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.To correct for volume loss during pipetting, increase the cDNA hybridization mix volume by 10 to 15% with equal parts Enhanced Hybridization Buffer and water (see Appendix C for recommended hybridization volumes for various size coverslips).

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