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800-985-CBMX • 425-493-2000
http://www.combimatrix.com

What is the principle behind the CombiMatrix CustomArray™ Stripping Kit?
How are the labeled targets stripped from CombiMatrix microarrays?
What types of labeled targets can be stripped from CombiMatrix microarrays?
What types of CombiMatrix microarrays can be stripped?
Can I strip CombiMatrix microarrays that have long oligonucleotide probes over 40-mer?
How many times can a CombiMatrix microarray be stripped and re-hybridized?
Can I strip CombiMatrix microarrays hybridized a long time ago?
Can I strip CombiMatrix microarrays if they have dried after hybridization?
Can third party arrays be stripped using the CombiMatrix stripping protocol?
Can I use some alternative protocols for stripping of CombiMatrix microarrays?

What is supplied in the CustomArray™ Stripping Kit?
What special equipment do I need to use with the CustomArray™ Stripping Kit? Do I need to purchase additional items from CombiMatrix to strip microarrays?
How many times can I re-use the Stripping Cap provided with the CustomArray™ Stripping Kit?
How many times can I re-use the CustomArray™ Stripping Clamp?
What method do I use for quality control of CombiMatrix microarrays after stripping?
What scanners do I use?
What am I seeing on my scanned image after stripping?

How long does it take to strip a CombiMatrix microarray using the CustomArray™ Stripping Kit?
How long can I store hybridized CombiMatrix microarrays prior to stripping?
Can I use a CombiMatrix microarray more than four times?
Do I need to rotate CombiMatrix microarrays during stripping?
What happens if I introduce air bubbles into the stripping chamber?

What protocol can I use to re-hybridize stripped CombiMatrix microarrays?
Do I need to perform re-hydration and pre-hybridization steps with stripped CombiMatrix microarrays?
Can I change conditions between subsequent hybridizations of the same CombiMatrix microarray?
Can I change the type of labeled targets between subsequent hybridization of the same CombiMatrix microarray?
Do I need to increase the amount of labeled targets for subsequent re-hybridizations of the stripped CombiMatrix microarray?
How do I get a Hybridization Cap for re-hybridization of stripped CombiMatrix microarrays? Can I use the Hybridization Cap shipped with my original CombiMatrix microarray for re-hybridization?

How do I store stripped CombiMatrix microarrays?
What is the shelf life of a stripped CombiMatrix microarrays? How long can I store CombiMatrix microarrays after stripping?
Can I dry stripped CombiMatrix microarrays?

Will signal intensity decrease during subsequent stripping and re-hybridization steps?
How reproducible are CombiMatrix microarrays after stripping and re-hybridization?
Can I strip CombiMatrix microarrays used for Hyb & Seq™ application?

WHAT IS THE PRINCIPLE BEHIND THE COMBIMATRIXCUSTOMARRAY™ STRIPPING KIT?
The CombiMatrix stripping protocol is based on chemical denaturation of DNA:DNA and DNA:RNA hybrids between oligonucleotide probes on microarrays and corresponding labeled targets. As a result of this denaturation, labeled targets are removed from oligonucleotide probes by washing. Both stripping and subsequent washing are performed using very stringent conditions to ensure complete target removal. The CombiMatrix microarray platform is the only one on the market that can withstand this type of chemical treatment. CombiMatrix recommends performing stripping three times. Studies have shown that stripping more than three times affects the microarray quality. In total, the CombiMatrix stripping protocol with the CustomArray™ Stripping Kit enables the use of a single microarray four times. The CustomArray™ Stripping Kit has been optimized for DNA and RNA targets labeled with biotin, and with fluorescent dyes Cy5 and AlexaFluor®. Use of the CustomArray™ Stripping Kit for targets labeled with other fluorescent dyes (such as Cy3) is not recommended. Please note that the protocol is designed for microarrays that have standard oligonucleotide probes of 40-mer or less.

Hybridization of CombiMatrix microarrays can be successfully performed using a wide variety of techniques for target preparation and labeling. However, some of these experimental approaches can result in irreversible target binding and thus preclude microarray stripping. CombiMatrix recommends the investigators to test whether their experimental approach is suitable for microarray re-use.

HOW ARE THE LABELED TARGETS STRIPPED FROM COMBIMATRIX MICROARRAYS?
The CustomArray™ Stripping Kit is designed to provide reagents for stripping and preparation of microarrays for subsequent re-use. The labeled targets are stripped as a result of chemical denaturation of DNA:DNA and DNA:RNA hybrids. This denaturation is performed under very stringent conditions to ensure complete target removal. Please note that the protocol is designed for CombiMatrix microarrays that have standard oligonucleotide probe length of 40-mer or less.

WHAT TYPES OF LABELED TARGETS CAN BE STRIPPED FROM COMBIMATRIX MICROARRAYS?
The CustomArray™ Stripping Kit has been optimized for DNA and RNA targets labeled with biotin, and with fluorescent dyes Cy5 and AlexaFluor® 647.

Use of the CustomArray™ Stripping Kit for targets labeled with other fluorescent dyes is not recommended. Specifically, Cy3® labeled probes may exhibit residual background after stripping. The stripping protocol is recommended for CombiMatrix microarrays that have standard oligonucleotide probe length of 40-mer or less, longer probes may not be stripped completely. Effective stripping also depends on the user's selection of target labeling and preparation protocols, and microarray handling during hybridization. CombiMatrix recommends the investigators to test whether their experimental approach is suitable for microarray re-use. The following issues may result in incomplete stripping:

• Drying of the semiconductor surface while targets are hybridized to microarray probes will invariably preclude stripping of the microarray. Once the hybridized nucleic acids are dried, they cannot be removed.
• Labeling of targets with incorporation of amino allyl nucleotide derivatives. CombiMatrix does not recommend amino-allyl labeling, instead use the labeling methods for direct dye or biotin incorporation.
• Nucleic acid targets of large size tend to precipitate in the porous reaction layer covering the microarrays semiconductor and will not strip easily. Low molecular weight target material must be used for stripping to be effective. To achieve optimum hybridization and stripping results, target samples need to be fragmented. The fragment size should be in the range of 50 to 200 nucleotide for RNA, and from 50 to 500 nucleotide for DNA, respectively.
• Microarray overloading with targets during hybridization. CombiMatrix recommends to use no more than 5 microgram for a 12K microarray (or 2 microgram for a sector of a 4X2K microarray). In some cases, saturated (i.e. overloaded) spots cannot be completely stripped, and then the concentration of target material should be reduced for efficient stripping.

WHAT TYPES OF COMBIMATRIX MICROARRAYS CAN BE STRIPPED?
The following types of CombiMatrix microarrays can be successfully stripped using the CustomArray™ Stripping Kit:

• CustomArray™ 12K and 4x2K microarrays providing that they have standard oligonucleotide probe length (40-mer or less);
• Species Specific and Content Choice MicroRNA 4X2K Microarrays;
• CatalogArrays based on both 12K and 4x2K formats.

CAN I STRIP COMBIMATRIX MICROARRAYS THAT HAVE LONG OLIGONUCLEOTIDE PROBES (OVER 40-MER)?
No, the current Stripping Protocol is designed for CombiMatrix microarrays that have standard oligonucleotide probes of 40-mer or less. Probes that are longer than 40-mer may not be stripped completely.

HOW MANY TIMES CAN A COMBIMATRIX MICROARRAY BE STRIPPED AND RE-HYBRIDIZED?
Stripping and re-hybridization may be repeated three times, therefore each CombiMatrix microarray can be used up to a maximum of four times. CombiMatrix does not recommend increasing the number of re-uses. Please note that some loss of quality may be observed during re-use.

CAN I STRIP COMBIMATRIX MICROARRAYS HYBRIDIZED A LONG TIME AGO?
Hybridized CombiMatrix microarrays may be stripped and re-used only providing that they are kept wet, either in a tube or slide-holder containing 1xPBS, or in Imaging Solution with the LifterSlip™ attached. Do not allow the semiconductor microarray surface to dry, as this will prevent any further use. Avoid prolonged storage of hybridized arrays prior to stripping: instead, strip the microarrays first, and store them wet in Imaging Solution or 1xPBS at 4oC for a maximum of 2 weeks.

CAN I STRIP COMBIMATRIX MICROARRAYS IF THEY HAVE DRIED AFTER HYBRIDIZATION?
No, if CombiMatrix microarrays become dry at any step after hybridization, the product is not longer usable for stripping and re-hybridization.

CAN THIRD PARTY ARRAYS BE STRIPPED USING THE CUSTOMARRAY™ STRIPPING PROTOCOL?
The CombiMatrix striping protocol is intended for use exclusively with CombiMatrix microarrays. Both stripping and subsequent washing are performed under very stringent conditions to ensure complete target removal. The CombiMatrix microarray platform is the only one that can withstand this type of chemical treatment.

CAN I USE SOME ALTERNATIVE PROTOCOLS FOR STRIPPING OF COMBIMATRIX MICROARRAYS?
CombiMatrix tried several alternative protocols, all of them provided insufficient stripping of targets. Even in the cases when no hybridization signal could be observed after stripping, some residual nucleic acid hybrids remained on microarrays and adversely affected the results of subsequent re-hybridizations. Thus, usage of the recommended protocol with the CustomArray™ Stripping Kit is the only means to preserve integrity of CombiMatrix microarrays.

WHAT IS SUPPLIED IN THE CUSTOMARRAY™ STRIPPING KIT?
One kit is intended for 25 uses. The Kit includes new hybridization caps, thus, the user must specify the format of microarrays that will be subjected to stripping (12K or 4X2K).

WHAT SPECIAL EQUIPMENT DO I NEED TO USE WITH THE STRIPPING KIT? DO I NEED TO PURCHASE ADDITIONAL ITEMS FROM COMBIMATRIX TO STRIP COMBIMATRIX MICROARRAYS?
The CustomArray™ Stripping Clamp is needed to assemble and fasten together the microarray and the Stripping Cap. Do NOT use the CustomArray™ 12K or 4x2K Hybridization Clamp with this kit.

HOW MANY TIMES CAN I RE-USE THE STRIPPING CAP PROVIDED WITH THE CUSTOMARRAY™ STRIPPING KIT?
The same Stripping Cap and screw plugs can be used for all 25 stripping reactions provided in the Kit. Wash the Stripping Cap and plugs between uses (with ethanol) and change the gaskets (O-rings). The disposable gaskets supplied with the kit are intended for single-use only. Re-use of gaskets may cause leakage of the Stripping Solution from the Stripping Cap. Dispose of the used gasket upon completion of this protocol.

HOW MANY TIMES CAN I RE-USE THE COMBIMATRIX STRIPPING CLAMP?
The CustomArray™ Stripping Clamp may be used up to 100 times. Some staining may occur on the surface of Clamp from spilled Stripping Solution, however, this will not affect the Clamp performance. Wash the Clamp with ethanol between uses.

WHAT METHOD DO I USE FOR QUALITY CONTROL OF CUSTOMARRAY™ MICROARRAYS AFTER STRIPPING?
The quality of stripping must be determined prior to re-hybridization. This is achieved by imaging the stripped microarray with the same high-resolution fluorescent microarray scanner and the same PMT gain settings as used for the first hybridization and imaging of the microarray. Do not increase the PMT gain setting, as this may increase the background fluorescence.

After you complete the scan, you can examine the resultant image by eye and/or perform data extraction. The stripping reaction has been successful if there are no spots visible on the array. You may still see some elements of the semiconductor array, such as the grid and contact pads along the side (Fig. 1, compare images before (A) and after stripping (B)).

Figure 1. Images of a bottom sector of a CustomArray™ 4x2K microarray before (A) and after (B) stripping.

WHAT SCANNERS DO I USE?
CombiMatrix microarrays are compatible with a number of imaging systems that have been developed for spotted microarrays. CombiMatrix recommends a slide-based, focusable scanner with a minimum of 5 micron resolution. Axon, Perkin Elmer, and BioRad all have models compatible with our chips (Axon GenePix 4000B, GenePix 4200A, GenePix 4200AL; Perkin Elmer ScanArray™ 4000 and 5000, ScanArray™ Lite, ScanArray™ Express). Contact a technical support representative at CombiMatrix with any questions regarding the compatibility of the imaging system that is being utilized.

WHAT AM I SEEING ON MY SCANNED IMAGE AFTER STRIPPING?
The stripping reaction has been successful if there are no spots visible on the array. You may still see some elements of the semiconductor array, such as the grid and contact pads along the side (Fig. 1, compare images before (A) and after stripping (B)). When scanning the stripped microarray, use the SAME scanner PMT gain setting as was used for the original hybridization and imaging. Do not increase the PMT gain setting, as this may increase the background fluorescence.

HOW LONG DOES IT TAKE TO STRIP COMBIMATRIX MICROARRAYS USING THE STRIPPING KIT?
CombiMatrix microarray stripping, washing and subsequent quality checking by imaging take approximately 1.5 to 2 hours.

HOW LONG CAN I STORE COMBIMATRIX MICROARRAYS PRIOR TO STRIPPING?
Hybridized CombiMatrix microarrays may be stored for a short time either in a tube or slide-holder containing 1xPBS, or in Imaging Solution with the LifterSlip™ attached. Avoid prolonged storage of hybridized arrays prior to stripping. CombiMatrix recommends performing the stripping reaction immediately after a good image of a hybridized array is obtained. Do not allow the semiconductor microarray surface to dry as this will prevent any further use.

CAN I USE A COMBIMATRIX MICROARRAY MORE THAN FOUR TIMES?
CombiMatrix recommends performing stripping three times. Studies have shown that stripping more than three times affects the microarray quality. In total, the CombiMatrix stripping protocol with the CustomArray™ Stripping Kit enables the use of a single CombiMatrix microarray four times.

DO I NEED TO ROTATE COMBIMATRIX MICROARRAYS DURING STRIPPING?
The assembled Stripping Clamp with microarray should not be rotated during the stripping incubation. Lay the CustomArray™ Stripping Clamp assembly horizontally to keep the air bubble(s) at the top of the chamber and incubate it at 65°C.

WHAT HAPPENS IF I INTRODUCE AIR BUBBLES INTO THE STRIPPING CHAMBER?
Air bubbles may form when you fill the stripping chamber. However, they will not interfere with stripping due to the large volume of the chamber (500 μl). Lay the Stripping Clamp assembly horizontally to keep the air bubble(s) at the top of the chamber during the incubation steps.

WHAT PROTOCOL CAN I USE TO RE-HYBRIDIZE COMBIMATRIX MICROARRAYS?
Follow the corresponding CombiMatrix Hybridization Protocol recommended for your microarray format and application. You may change the hybridization conditions if you wish, but you must perform the pre-hybridization steps as described in the protocol.

DO I NEED TO PERFORM RE-HYDRATION AND PRE-HYBRIDIZATION STEPS WITH STRIPPED COMBIMATRIX MICROARRAYS?
Yes, it is important that the pre-hybridization steps are completed as described in the hybridization protocol for CombiMatrix microarrays.

CAN I CHANGE CONDITIONS BETWEEN SUBSEQUENT HYBRIDIZATIONS OF THE SAME COMBIMATRIX MICROARRAY?
Yes, you may change the hybridization conditions between subsequent re-hybridizations of the same CombiMatrix microarray.

CAN I CHANGE THE TYPE OF LABELED TARGETS BETWEEN SUBSEQUENT HYBRIDIZATIONS OF THE SAME COMBIMATRIX MICROARRAY?
Yes, different types of labeled targets can be used for subsequent re-hybridizations of the same CombiMatrix microarray. The Stripping Kit, however, has been optimized for DNA and RNA targets labeled with Cy5 and AlexaFluor® 647 fluorescent dyes.

DO I NEED TO INCREASE THE AMOUNT OF LABELED TARGETS FOR SUBSEQUENT RE-HYBRIDIZATIONS OF STRIPPED COMBIMATRIX MICROARRAYS?
No, you can use the same amount of labeled targets for each subsequent re-hybridization.

HOW DO I GET A HYBRIDIZATION CAP FOR RE-HYBRIDIZATION OF A STRIPPED COMBIMATRIX MICROARRAY? CAN I USE THE HYBRIDIZATION CAP SHIPPED WITH MY ORIGINAL MICROARRAY FOR RE-HYBRIDIZATION?
The CustomArray™ Stripping Kit contains new hybridization caps (plus plastic clips for the 12K format) for hybridization of the stripped microarrays. Do NOT re-use hybridization caps (and plastic clips), discard them upon completion of the hybridization protocol. When you order the Stripping Kit, you must specify the format of microarrays that will be subjected to stripping (12K or 4X2K).

HOW DO I STORE STRIPPED COMBIMATRIX MICROARRAYS?
After imaging of the stripped CombiMatrix microarray keep the LifterSlip™ coverslip on the array surface. Add extra imaging solution to prevent the microarray from drying. Cover the microarray and LifterSlip™ with plastic wrap to prevent evaporation. Alternatively, the stripped microarray can be stored without a LifterSlip™ in a tube or slide holder containing 1xPBS. Store at 4oC to prevent bacterial growth. Stripped microarrays can be stored for 2 weeks in this manner.

WHAT IS THE SHELF LIFE OF A STRIPPED COMBIMATRIX MICROARRAY? HOW LONG CAN I STORE COMBIMATRIX MICROARRAYS AFTER STRIPPING?
Avoid long-term storage of stripped CombiMatrix microarrays. Microarrays should be re-used within two of weeks from the time of stripping. The stripped CombiMatrix microarray must be kept wet, either in 1xPBS or in the Imaging Solution with the LifterSlip™ attached. If the array dries out it cannot be re-used.

CAN I DRY STRIPPED COMBIMATRIX MICROARRAYS?
No, if a stripped CombiMatrix microarray dries out, it can NOT be re-used.

WILL SIGNAL INTENSITY DECREASE DURING SUBSEQUENT STRIPPING AND RE-HYBRIDIZATION STEPS?
CombiMatrix hybridized several sectored CustomArray™ 4x2K chips with the same labeled RNA target sample prepared from the Universal Human Reference RNA (Stratagene). Then the microarrays underwent 3 stripping and re-hybridization steps with the same labeled target sample. Typical images of subsequent re-hybridizations are shown in Fig. 2, demonstrating that the average signal intensity does not decrease during re-usage. However, additional stripping steps exceeding the 3-time limit may adversely affect spot morphology and data quality.

Figure 2. Images of a sector of a CustomArray™ 4x2K microarray obtained at subsequent re-hybridization steps. The left panel corresponds to the 1st hybridization, and the right panel to the 4th hybridization, respectively.

HOW REPRODUCIBLE ARE COMBIMATRIX MICROARRAYS AFTER STRIPPING AND RE-HYBRIDIZATION?
CombiMatrix hybridized several sectored CustomArray™ 4x2K chips with the same labeled RNA target sample prepared from the Universal Human Reference RNA (Stratagene). Then the microarrays underwent 3 stripping and re-hybridization steps with the same labeled target sample. The raw data (without normalization and background correction) were log-transformed, and pair-wise correlation coefficients (r2) were calculated for subsequent re-hybridizations. The average r2 was 0.91. In total, approximately 80% of all correlation coefficients exceeded 0.9. Scatter plots of log-transformed data are shown in Fig. 3 demonstrating high consistency between four subsequent hybridizations. Please note that the 5th, additional hybridization of the same microarray results in a decrease in data quality (the bottom panel, right plot, comparison of 1st hybridization with the 5th one for the same sector).

Figure 3. Scatter plots of data obtained with sectored CustomArray™ 4x2K microarrays that were hybridized and re-hybridized with the same labeled RNA target sample prepared from the Universal Human Reference RNA (Stratagene). The microarrays underwent 3 standard stripping steps, and an additional 4th step. At the upper panel, X-axis is Log2 (Intensity from 1st hybridization), Y-axis is Log2 (Intensity from 2d hybridization) for each of 4 sectors of CombiMatrix microarray™ 4x2K. The lower panel compares Log2 (Intensity) from subsequent hybridizations of the same sector (sector C): 1st versus 2nd, 2nd versus 3rd, 3rd versus 4th, and 1st versus 5th, respectively. Log-transformed data are used without normalization and background correction.

CAN I STRIP COMBIMATRIX MICROARRAYS USED FOR HYB & SEQ™ APPLICATION?
No, during Hyb & Seq™ hybridization the labeled targets are covalently linked to the array probes and can not be stripped. Therefore, CombiMatrix microarrays can be used for Hyb & Seq™ only once.

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Date Last Modified: 05/02/2007