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FAQs : Microarrays

Real-time PCR and PCR Array Technical FAQ

A. Weak or No Signals

Low signal intensity is an indication of poor RNA starting material quality. RNA quality is crucial to the success of the array. Double-check the method of RNA isolation used as well as the quality and integrity of your experimental RNA as described. If the RNA has been prepared improperly or if there is any evidence of poor RNA quality, perform your RNA preparation again before repeating the array analysis.

Other than RNA sample quality, the following factors may also impair your results.

  1. Low RNA sample concentration.
    If the RNA sample is too dilute, it may not perform well in the assay. Concentrate your total RNA (to at least 11 ng/µl or up to 0.5 mg/ml) using a standard ammonium acetate ethanol precipitation based method.
  2. Inhibited cRNA Target Synthesis.
    1. Total RNA should be dissolved in RNase-free dH2O or RNase-free 10 mM Tris buffer pH 8.0. Do not use DEPC-treated water.
    2. If CsCl, LiCl, guanidinium, or organic extractions were used in the process of your total RNA purification, trace amounts of these contaminants will inhibit cRNA target synthesis. Clean up the RNA with our ArrayGrade™ Total RNA Isolation Kit (SABiosciences, Catalog # GA-013) to insure good target synthesis and good GEArray results.
  3. Improper washing in 0.1X SSC solution. Excessive washing of the membrane in the high stringency buffer (Wash Solution 2) will strip off the hybridized probes. Decrease the washing time.
  4. Improper hybridization temperature. Check the actual temperature inside your hybridization oven with a thermometer. The digital temperature reading on your hybridization oven could be several degrees off calibration. As the temperature goes higher than 60ºC the amount of specifically hybridized target will decrease until all targets and probes are denatured.
  5. Underexposure. Try multiple exposures for various longer times.

B. High Background

Other than RNA quality, the following factors may cause high background:

  1. Pre-hybridization step incomplete. Needs at least 30 minutes.
  2. Inaccurate AP-streptavidin dilution. Since the AP-streptavidin is dissolved in a glycerol containing solution, special caution should be taken when pipeting the small volume of AP-streptavidin. We suggest diluting no less than 2 µl of AP-streptavidin into 16 ml of Buffer Q (a 1:8,000 dilution). Draw up the needed volume, and carefully wipe the outside of the pipet tip with a laboratory wipe before dispensing the volume. The final working AP-streptavidin dilution can also be increased up to 1:12,000.
  3. Improper incubation time with AP-streptavidin. The incubation should be no longer than 10 minutes, but it may also be reduced.
  4. Improper washing temperature conditions. Be sure to use 2.0 ml Wash Solution 2 during the high-stringency washing step at 60ºC.
  5. Biotin contamination in containers or buffers. Milk contains a high amount of biotin. Never use any lab ware that has been used for Western blotting.
  6. Overexposure. Try multiple exposures for various shorter times.
  7. Too much total RNA or labeled cRNA target. Reduce the amount of total RNA or used for cRNA Target Synthesis and Labeling or reduce the amount of labeled cRNA used for hybridization.

C. I have performed the pre-hybridization of the array, but my cRNA synthesis efficiency is poor. Can I save the membrane until I try the assay again?
Yes. Rinse the GEArray with RNase-free water and allow to dry at room temperature. Once you define and correct the problem and repeat the RNA isolation and / or cRNA synthesis reaction, repeat the pre-hybridization of the array with fresh GEAprehyb pre-warmed to 60ºC.

D. Can I strip and re-probe the membranes?

The Oligo GEArray® is designed for one-time use only. Stripping and re-probing is not recommended. We found that stripping results by individual users varies widely, and even under the best conditions, 80 percent of the original signal is lost despite the UV-crosslinking of the oligonucleotides to the nylon membrane. Stripping and re-probing may be attempted under extreme circumstances or for troubleshooting purposes, but the subsequent results will not be publication quality.

Strip the membranes using a procedure similar to stripping blots used for Northern or Southern hybridization: Boil for 5 to 10 minutes in 0.5 % SDS, cool for 10 minutes, and rinse twice with 2X SSC. Store the damp membrane at -20 ºC in its original plastic tube if needed.