FAQ & Troubleshooting Guide
One ampliPHOX reagent kit (AP-5005) has sufficient reagents for 50 arrays.
We typically quote 4-6 weeks lead time when a new Custom Array order is placed. If you are reordering Custom Arrays, the lead time is generally 2-4 weeks. The time is primarily dependent on how quickly we have the captures on hand and scheduling a time for the arrays to be printed. Depending on your preference, we can order the capture oligos/proteins or you can order them and subsequently ship to us.
Depending on the total number of microarray spots, Custom Arrays are usually printed with the following spot size and center to center pitch unless requested otherwise when ordering:
|Number of Spots:||Spot Size||Pitch|
|Less Than 451||300 µm||400 µm|
|451 to 850||200 µm||300 µm|
|851 to 1900||100 µm||200 µm|
For Custom Arrays that will be used with ampliPHOX, the maximum array area is approximately 10.0 mm wide by 7.5 mm tall and can hold up to 850 unique captures. Additionally, we recommend including multiple replicates of each capture on your array to yield the best results statistically. Don’t forget that each array needs to incorporate at least 3 control spots to serve as a positive control and for mask alignment with the ampliVIEW software.
A focused microarray is just another term for low-density microarrays, they may also be called targeted microarrays. Low-density microarrays are typically less than 2500 spots per array while high-density micrarrays contain more more than 2500 spots. High-density microarrays can be used to evaluate an entire genome while low-density microarrays will focus on specific genes of interest.
Here’s a list of some frequently used terms and what they mean:
Capture – A capture is the material spotted onto the microarray. This can be an oligo, antibody, antigen, etc.
Target – A target is what will be detected by the microarray captures.
Bulk – Bulk is the excess polymer on a photoactivated array when the activation time is too long. In extreme cases the excess polymer will overtake the entire array.
Pitch – The pitch is the distance from the center of one microarray spot to the center of the adjacent spot.
Mask – In the ampliVIEW software, a mask is essentially the layout for your array. Using the software you can specify the location of each capture and control for easy array analysis.
Focused Microarray – A microarray in which there are less than 2500 spots. Also referred to as low-density or targeted microarrays.
The first line of defense against bulk polymerization is running the Full System Optimization or System/Reagent Check as outlined in the ampliPHOX Operation Manual to ensure you have the appropriate photoactivation time. There are many factors that can influence the photoactivation time including lot to lot variability, oxygen exposure, other atmospheric conditions, etc.
If your calibration arrays are acceptable, the following list provides some potential reasons for the bulk:
- Insufficient rinsing of excess ampliPHY after photoactivating. Once the array has been photoactivated, it is able to withstand more vigorous washing, so be sure to rinse thoroughly.
- Reagents are expired. Check the order summary included with your shipment for expiration dates.
- ampliPHY reagents were not brought to room temperature or vortexed thoroughly.
- ampliPHY vial was not completely closed between uses and was overexposed to oxygen. Use a new aliquot to see if that resolves the issue.
- Insufficient washing prior to photoactivation can leave residual salts on the array surface. These salts can act as “nucleation” sites causing non-specific polymer formation and bulk.
High background on an array is generally due to pre-ampliPHOX processing steps. Here are the most likely culprits of high background:
- Failure to block the array surface appropriately during the hybridization/binding steps.
- Failure to optimize the hybridization, binding, or wash steps.
- Allowing arrays to dry between wash steps. Even after the visual salt residues are removed background can remain.
- Well leaked during hybridization or labeling.
If you are seeing low signal across the entire array (control spots as well as target captures), the issue is likely one of the following:
- Poor calibration of photoactivation time or failure to perform appropriate System Optimization or Reagent Check prior to testing.
- Using expired reagents. Check the order summary included with your shipment for expiration dates.
- Over washing the array. The orbital shaker may be set at too high of a setting or when rinsing using a wash bottle the spray is directed at the array.
If you are seeing low signal only on the target captures (the control spots are strong), the issue is likely related to poor target binding. Here are some methods to troubleshoot the issue:
- Check the target product using another method to ensure the target is present and viable for binding.
- Increase the hybridization or binding time and/or temperature.
- Optimize the hybridization/binding buffer used for the array.
- For nucleic acid arrays, double check the capture and target sequences/binding pairs.
- For nucleic acid arrays in which a digest is being performed, optimize the time and concentration and ensure phosphorylation of the appropriate strand.
- For nucleic acid arrays in which a fragmentation is being performed, optimize the time, temperature and, if enzymatic, concentration.
Odd spot morphology can be caused by a number of reasons:
- When very strong signals are present and the array has not been adequately rinsed post-staining, bleading of the stain out of the spot can occur, leading to strangely shaped spots.
- Microarray printing is not always perfect. There is not always a uniform distribution of capture across the spot. This can result in some strange morphologies once photopolymerized and stained.
- “Comet-tail” spotting on all spots of the given target/concentration is indicative of the spotting concentration being too high during printing.
These anomalies are referred to as array artifacts and are not unique to ampliPHOX. Here’s what you may be seeing:
- “Hollow” Spots: These are areas where a capture has been spotted but the target is not present in the sample so the result is negative. However, due to the change in surface properties caused by the binding of the capture, the local background signal is higher than than the negative spot signal.
- Random Spots: These are likely due to residual salts or dust particles. Be sure to wash your arrays well.
- Streaks or Wipe Marks: This is likely damage to the array surface, usually caused by a pipette tip or tissue wipe during processing.
If you can’t find the answer to your question please call 303-402-9100, 8:00 am to 5:00 pm MST or e-mail firstname.lastname@example.org.