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
- 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.
- Inhibited cRNA Target Synthesis.
- 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.
- 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.
- 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.
- 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.
- Underexposure. Try multiple exposures for various longer times.
B. High Background
Other than RNA quality, the following factors may cause high background:
- Pre-hybridization step incomplete. Needs at least 30 minutes.
- 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.
- Improper incubation time with AP-streptavidin. The incubation should be no
longer than 10 minutes, but it may also be reduced.
- Improper washing temperature conditions. Be sure to use 2.0 ml Wash
Solution 2 during the high-stringency washing step at 60ºC.
- 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.
- Overexposure. Try multiple exposures for various shorter times.
- 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
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
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.