Products - DNA Microarrays - CheckIt™ Chips Microarrays to Test Microarray Hybridization, Processing, Scanning and Quantification for Research and Educational Purposes

Microarrays and fluorescent probes for test hybridization and quality control experiments. Excellent for product development and educational purposes to benchmark microarray hybridization, processing, scanning and data quantification.

Table of Contents

• Introduction
• Quality Control
• Product Description
• Kit Applications
• Technical Assistance
• Protocols (1-3)
• Content Map (Table 1)
• Troubleshooting Tips
• Recommended Products
• Ordering Information
• Warranty

Introduction
Congratulations on taking a big step towards improving your microarray research. This booklet contains complete protocols outlining the steps required to use CheckIt™ Chips for quality control and test hybridization experiments. An excellent introductory product for users new to the field, and must-have product for advanced users wishing to check their hybridization probes prior to use on expensive, high-complexity microarrays.

Quality Control
Arrayit assures the performance of these products, and the finest scientific research went into their development. The SeeIt™ Universal Probe Solution in CheckIt™ Chips Kits should be stored at 4ºC upon arrival. All other components should be stored at room temperature.

Product Description
ArrayIt® CheckIt™ Chips are manufactured in class 1 cleanrooms using the most sophisticated robotics, surfaces, reagents, and protocols available. This exacting attention to detail ensures optimal product performance, including highly stable attachment of target sequences, efficient hybridization, and easy detection and quantification. CheckIt™ Chips kits contains the following components:

• Five (5) CheckIt™ Chips microarrays (25 mm x 76 mm) printed with 200 oligonucleotides targets (70-mers) in 300 µm spots, and configured as two identical 10 x 10 subgrids spaced 4.5 mm apart.
• 1.0 ml of 1.25X SuperHyb™ Hybridization Solution
• 50 µl of 5X SeeIt™ Universal Probe Solution (50 µM Cy3-labeled SeeIt™ Universal Probe)
• Eight (8) glass coverslips (22 X 22 mm)

ArrayIt® CheckIt™ Chips are designed for test hybridizations and quality control experiments for printed microarrays and labeled probes. Users will appreciate the following kit features:

• Manufactured in advanced class 1 cleanrooms
• Optically flat SuperAmine substrates
• Stable attachment of target elements to glass surface
• 70-mer oligonucleotide targets from human, mouse and E. coli
• 300 µm diameter spots are easy to detect
• Universal SeeIt™ Probe will hybridize to any nucleic acid microarray
• Ultra-pure reagents used for all kit components
• Check quality of microarrays and probes made “in house”
• Marker spots permit rapid spot finding and quantitation
• Inexpensive introduction to microarray analysis
• Excellent for beginning, intermediate or advanced users

Kit Applications
Uses for the CheckIt™ Chips Kits include:
1. Practice microarray experiments for beginning microarray users. Kits teach beginners how to process, hybridize, wash, detect and quantitate microarrays. Protocol #1 allows beginners to obtain high-quality microarray data in one hour.

2. Check the quality of nucleic acid microarrays purchased from other vendors or made “in-house”. Hybridization with the SeeIt Universal Hybridization Probe, a Cy3-labeled mixture of SeeIt™ Universal Probe, tests for the presence of target sequences on any nucleic acid microarray. Protocol #2 allows intermediate and advanced users to perform quality control (QC) on microarrays prior to actual experimentation.

3. Check the quality of any human or mouse fluorescent cDNA mixture by hybridizing labeled mixtures to CheckIt™ Chips, which contain 70-mers complementary to human and mouse mRNAs. Protocol #3 allows intermediate and advanced users to perform quality control (QC) on labeled probes prior to use on high-complexity genomic microarrays.  CheckIt™ Chips also allow the testing of stringency, wash protocols and different hybridization buffers. The presence of sense and antisense target elements allow hybridization to labeled sense and antisense strands.

Technical Assistance
Please contact us if you have any comments, suggestions, or if you need technical assistance. By electronic mail: arrayit@arrayit.com (under the subject heading, please type ArrayIt® technical assistance). By telephone: (408) 744-1331, Monday–Friday PST 8:30am - 5:30 PM. Please remember that we want to hear about your successes!

Protocol 1:  Practice microarray experiments for beginning users.
Short Protocol #1 (steps 1-5)
1. Process CheckIt™ Chips to remove unbound target sequences.
2. Hybridize CheckIt™ Chips to allow binding of SeeIt™ Universal Probe.
3. Wash microarrays to remove unbound SeeIt™ Universal Probe.
4. Scan or image the microarrays to obtain data (.tif) files.
5. Quantitate data.

Complete Protocol #1 (steps 1-5)
1. Process CheckIt™ Chips to remove unbound target sequences. Hybridization efficiency and data quality are improved by processing CheckIt™ Chips prior to hybridization. CheckIt™ Chips kit are printed on SuperAmine Microarray Substrates and coupled to the surface by baking for 80 min at 80°C in a drying oven. CheckIt™ Chips are shipped unprocessed. Prior to processing, mark the location of the microarray by scoring the UNDERSIDE (the side that is NOT bar-coded) of the microarray gently with a diamond pencil.  Printed microarrays are located approximately 10 mm from the top edge and 10 mm from the left edge of the substrate.  The two identical subgrids are spaced 4.5 mm apart and contain 200 spots 300 µm in diameter.  Spot are visible to the naked eye prior to processing, but not afterwards. Washing and drying can be expedited using the ArrayIt® High Throughput Wash Station and Microarray High Speed Centrifuge or similar devices.  Follow the 6-step protocol to process CheckIt™ Chips.

A. Gently score the underside of the substrate to define the microarray (optional).
B. Wash for 2 minutes at 20-25°C in 1X Wash Buffer 1.
C. Wash for 2 minutes at 20-25°C in 1X Wash Buffer 2.
D. Treat microarrays for 2 minutes at 100ºC in dH2O. Cool to room temperature.
E. Fix microarrays for 2 minutes in ice cold 100% ethanol.
F. Spin microarrays dry by centrifugation.

2. Hybridize CheckIt™ Chips to allow binding of the SeeIt™ Universal Probe.Mix 2.0 µl of 5X SeeIt™ Universal Probe (50 µM) with 8.0 µl of 1.25X SuperHyb™ Hybridization Buffer in a microfuge tube to provide 10.0 µl of 1X probe solution (10 µM final). Mix contents thoroughly by tapping the tube or by gentle vortexing. Apply the 10.0 µl 1X probe mixture onto the top (bar-coded) surface of the microarray adjacent to the printed microarray near the top edge of the substrate. The top edge should contain a cropped corner (chamfer) as the right corner. Place a glass cover slip (22 x 22) onto the 10.0 µl probe droplet and lower it gently onto the microarray surface making sure not to trap air bubbles under the cover slip. Place the SeeIt™ Chip containing the probe solution and cover slip into a Hybridization Cassette or a similar device, and incubate at room temperature (20-25ºC) for 30 minutes. Make sure to use 10 µl of dH2O in the Hybridization Cassette to prevent probe dessication during the 30 minute reaction. The cassette can also be placed in a water bath to ensure precise temperature control during the hybridization.

3. Wash microarrays to remove unbound SeeIt™ Universal Probe. After the 30 minute hybridization reaction, remove the microarray from the Hybridization Cassette and move the microarray immediately into the wash steps.  The faster the better at this stage.  If the fluorescent probe dries onto the microarray surface, the background signal will be elevated, complicated the detection and quantitation procedures. Do not allow the fluorescent sample to dry onto the substrate. The cover slip should detach in the first wash step within 30 seconds if it is hydrated properly. The ArrayIt® High Throughput Wash Station or similar device can be used for the wash steps, and the High Speed Microarray Centrifuge, Savant SpeedVac, or similar device can be used to dry the microarrays. See http://arrayit.com/PDF/ to download the details of these protocols.
The wash steps are performed using a 4-step procedure:
A. Wash the microarray for 1.5 minutes with 1X Wash Buffer 1.
B. Wash for 1 minute in 1X Wash Buffer 2.
C. Wash for 10 seconds in 1X Wash Buffer 3.
D. Spin the microarray dry by centrifugation.

4. Scan or image the microarrays to obtain data (.tif) files. Once the microarrays are washed and dried, place a CheckIt™ Chip into the scanner or imager and adjust the hardware settings to obtain strong fluorescent signals and low background. The two subgrids are located in ~1.0 cm2 area and the spots are 300 µm in diameter.  A scanner or imager resolution of 10 µm will provide an image of sufficient resolution.  Save the microarray image as a TIIFF (.tif) file. Scanners and imagers are provided by a number of commercial vendors including ArrayIt®, BioRad, PerkinElmer, Axon, Applied Precision and others.  Save the fluorescent image as a TIFF (.tif) file. Consult the user manual prior to using any microarray detection instrument.

5. Quantitate data. Open the .tif file and place a quantitation template over the microarray image.  Make sure that the template is placed accurately over the spots.  Use the “drag tools” to make minor adjustments to template location. Use the quantitation software to acquire the values located inside each template location.  Save the results as a comma- or tab-delimited file.  Delimited data can be imported into Spreadsheet and data mining tools and combined with content map information (see below).  Quantitation packages are provided with many commercial scanners and imagers, and can also be purchased as separate packages from a number of commercial sources including BioDiscovery, Inc (Marina del Ray, CA).

Protocol 2:  Allows intermediate and advanced users to assess the quality of any printed microarray.
Short Protocol #2  (steps 1-6)
1. Obtain preprinted microarrays.
2. Process microarrays and CheckIt™ Chips to remove unbound target sequences.
3. Hybridize microarrays to allow SeeIt™ Universal Probe to bind to target sequences.
4. Wash microarrays to remove unbound SeeIt™ Universal Probe.
5. Scan or image the microarrays to obtain data (.tif) files.
6. Quantitate data.

Complete Protocol #2 (steps 1-6)
1. Obtain preprinted microarrays.  Preprinted microarrays can be obtained from a commercial vendor or made in your own laboratory. When manufacturing your own microarrays, ArrayIt® brand SuperAmine or SuperAldehyde Substrates are recommended, though the components of CheckIt™ Chips Kits are compatible with microarrays printed on most other surfaces.

2. Process the microarrays and/or CheckIt™ Chips to remove unbound target sequences.  Hybridization efficiency and data quality are improved by processing microarrays to remove unbound sequences. CheckIt™ Chips kit are printed on SuperAmine Microarray Substrates and coupling is performed by baking for 80 min at 80°C in a drying oven.  CheckIt™ Chips are shipped unprocessed.  Prior to processing, users may want to mark the location of the microarray by scoring the UNDERSIDE (the side that is NOT bar-coded) of the microarray gently with a diamond pencil.  Printed microarrays are located approximately 10 mm from the top edge and 10 mm from the left edge of the substrate. The two identical subgrids are spaced 4.5 mm apart and contain 200 spots 300 µm in diameter.  Spot are visible to the naked eye prior to processing, but not afterwards.  Washing and drying can be expedited using the ArrayIt® High Throughput Wash Station and Microarray High Speed Centrifuge or similar devices. See http://arrayit.com/PDF to download the detailed and printable versions of all ArrayIt® brand Product protocols.  Follow the 6-step protocol to process your own microarrays and/or CheckIt™ Chips:
A. Gently score the underside of the substrate to define the microarray (optional).
B. Wash  for 2 minutes at 20-25°C in 1X Wash Buffer 1.
C. Wash  for 2 minutes at 20-25°C in 1X Wash Buffer 2.
D. Treat microarrays for 2 minutes at 100ºC in dH2O. Cool to room temperature.
E. Fix microarrays for 2 minutes in ice cold 100% ethanol.
F. Spin microarrays dry by centrifugation.

3. Hybridize CheckIt™ Chips to allow binding of the SeeIt™ Universal Probe. Mix 2.0 µl  of 5X SeeIt™ Universal Probe (50 µM) with 8.0 µl of 1.25X SuperHyb™ Hybridization Buffer in a microfuge tube to provide 10.0 µl of 1X probe solution (10 µM final).  Mix contents thoroughly by tapping the tube or by gentle vortexing. Apply the 10.0 µl 1X probe mixture onto the top (bar-coded) surface of the microarray adjacent to the printed microarray near the top edge of the substrate.  The top edge should contain a cropped corner (chamfer) as the right corner. Place a glass cover slip (22 x 22) onto the 10.0 µl probe droplet and lower it gently onto the microarray surface making sure not to trap air bubbles under the cover slip. Place the microarray or CheckIt™ Chip containing the probe solution and cover slip into a Hybridization Cassette or a similar device, and incubate at room temperature (20-25oC) for 30 minutes. Make sure to use 10 µl of dH2O in the Hybridization Cassette to prevent probe dessication during the 30 minute reaction. The cassette can also be placed in a water bath to ensure precise temperature control during the hybridization.

4. Wash microarrays to remove the unbound SeeIt™ Universal Probe.After the 30 minute hybridization reaction, remove the microarray from the Hybridization Cassette and move the microarray immediately into the wash steps. The faster the better at this stage. If the fluorescent probe dries onto the microarray surface, the background signal will be elevated, complicating the detection and quantitation procedures. Do not allow the fluorescent sample to dry onto the substrate. The cover slip should detach in the first wash step within 30 seconds if it is hydrated properly. The ArrayIt® High Throughput Wash Station or similar device can be used for the wash steps, and the High Speed Microarray Centrifuge, Savant SpeedVac, or similar device can be used to dry the microarrays. See http://arrayit.com/PDF/ to download the details of these protocols.
The wash steps are performed using a 4-step procedure:
A. Wash the microarray for 1.5 minutes with 1X Wash Buffer 1.
B. Wash for 1 minute in 1X Wash Buffer 2.
C. Wash for 10 seconds in 1X Wash Buffer 3.
D. Spin the microarray dry by centrifugation.

5. Scan or image the microarrays to obtain data (.tif) files. Once the microarrays are washed and dried, place a microarray or CheckIt™ Chip into the scanner or imager and adjust the hardware settings to obtain strong fluorescent signals and low background.  The two subgrids are located in a ~1.0 cm2 area and the spots are 300 µm in diameter.  A scanner or imager resolution of 10 µm will provide an image of sufficient resolution.  Save the microarray image as a TIIFF (.tif) file.  Scanners and imagers are provided by a number of commercial vendors including Virtek, Packard BioScience, Axon, Applied Precision and others.  Save the fluorescent image as a TIFF (.tif) file. Consult the user manual prior to using any microarray detection instrument.

6. Quantitate data. Open the .tif file and place a quantitation template over the microarray image.  Make sure that the template is placed accurately over the spots. Use the “drag tools” to make minor adjustments to template location. Use the quantitation software to acquire the values located inside each template location. Save the results as a comma- or tab-delimited file.  Delimited data can be imported into Spreadsheet and data mining tools and combined with content map information (see below). Quantitation packages are provided with many commercial scanners and imagers, and can also be purchased as separate packages from a number of commercial sources including BioDiscovery, Inc (Marina del Ray, CA).

Protocol 3:  Allows intermediate and advanced users to assess the quality of human and mouse fluorescent probe mixtures.
Short Protocol #3 (steps 1-6)
1. Prepare a fluorescent probe mixture from human or mouse.
2. Process CheckIt™ Chips to remove unbound target sequences.
3. Hybridize CheckIt™ Chips to allow binding of probe mixture and/or fluorescent 9-mers.
4. Wash microarrays to remove unbound probe molecules and/or 9-mers.
5. Scan or image the microarrays to obtain data (.tif) files.
6. Quantitate data.

Complete Protocol #3 (steps 1-6)
1. Prepare a fluorescent probe mixture from human or mouse. CheckIt™ Chips contain 70-mers corresponding to the coding and non-coding strands of human and mouse mRNAs, and these target sequences will hybridized to fluorescent mixtures prepared from mRNA or DNA from human and mouse.  Dozens of different labeling procedures are available for use with CheckIt™ Chips, including methods that use oligo-dT to prime total RNA or mRNA. After the labeling reaction, probe purification is recommended strongly, to remove contaminants such as unincorporated nucleotides, salts and proteins that will elevated background fluorescence. Probe purification can be accomplished using the ArrayIt® Fluorescent Probe Purification Kits or another suitable purification system.  CheckIt™ Chips are compatible with most labeled and purified probes.  As a positive control, users may want to prepare a control mixture provided with the CheckIt™ Chips kits. Mix 2.0 µl of 5X SeeIt™ Universal Probe (50 µM) with 8.0 µl of 1.25X SuperHyb™ Hybridization Buffer in a microfuge tube to provide 10.0 µl of 1X probe solution (10 µM final). Mix contents thoroughly by tapping the tube or by gentle vortexing.

2. Process the CheckIt™ Chips to remove unbound target sequences. Hybridization efficiency and data quality are improved by processing microarrays to remove unbound sequences. CheckIt™ Chips kit are printed on SuperAmine Microarray Substrates and coupling is performed by baking for 80 min at 80°C in a drying oven. CheckIt™ Chips are shipped unprocessed. Prior to processing, users may want to mark the location of the microarray by scoring the UNDERSIDE (the side that is NOT bar-coded) of the microarray gently with a diamond pencil.  Printed microarrays are located approximately 10 mm from the top edge and 10 mm from the left edge of the substrate.  The two identical subgrids are spaced 4.5 mm apart and contain 200 spots 300 µm in diameter.  Spot are visible to the naked eye prior to processing, but not afterwards. Washing and drying can be expedited using the ArrayIt® High Throughput Wash Station and Microarray High Speed Centrifuge or similar devices. See http://arrayit.com/PDF to download the detailed and printable versions of all ArrayIt® brand Product protocols.  Follow the 6-step protocol to process CheckIt™ Chips:
A. Gently score the underside of the substrate to define the microarray (optional).
B. Wash  for 2 minutes at 20-25°C in 1X Wash Buffer 1.
C. Wash  for 2 minutes at 20-25°C in 1X Wash Buffer 2.
D. Treat microarrays for 2 minutes at 100ºC in dH2O. Cool to room temperature.
E. Fix microarrays for 2 minutes in ice cold 100% ethanol.
F. Spin microarrays dry by centrifugation.

3. Hybridize CheckIt™ Chips to allow binding of fluorescent probe mixtures and/or control SeeIt™ Universal Probe.Apply the 10.0 µl of probe mixture onto the top (bar-coded) surface of the microarray adjacent to the printed microarray near the top edge of the substrate. The top edge should contain a cropped corner (chamfer) as the right corner.  Place a glass cover slip (22 x 22) onto the 10.0 µl probe droplet and lower it gently onto the microarray surface making sure not to trap air bubbles under the cover slip. Place the CheckIt™ Chip containing the probe solution and cover slip into a Hybridization Cassette or a similar device, and incubate at 65ºC (20-25°C for 9-mers) in a water bath for 30 minutes. Make sure to use 10 µl of dH2O in the Hybridization Cassette to prevent probe dessication during the 30 minute reaction.

4. Wash microarrays to remove unbound fluorescent probe molecules. After the 30 minute hybridization reaction, remove the microarray from the Hybridization Cassette and move the microarray immediately into the wash steps.  The faster the better at this stage.  If the fluorescent probe dries onto the microarray surface, the background signal will be elevated, complicating the detection and quantitation procedures.  Do not allow the fluorescent sample to dry onto the substrate.  The cover slip should detach in the first wash step within 30 seconds if it is hydrated properly.  The ArrayIt® High Throughput Wash Station or similar device can be used for the wash steps, and the High Speed Microarray Centrifuge, Savant SpeedVac, or similar device can be used to dry the microarrays.
The wash steps are performed using a 4-step procedure:
A. Wash the microarray for 1.5 minutes in 1X Wash Buffer 1.
B. Wash for 1 minute in 1X Wash Buffer 2.
C. Wash for 10 seconds in 1X Wash Buffer 3.
D. Spin the microarray dry by centrifugation.

5. Scan or image the microarrays to obtain data (.tif) files. Once the microarrays are washed and dried, place a CheckIt™ Chip into the scanner or imager and adjust the hardware settings to obtain strong fluorescent signals and low background. The two subgrids are located in a ~1.0 cm2 area and the spots are 300 µm in diameter. A scanner or imager resolution of 10 µm will provide an image of sufficient resolution.  Save the microarray image as a TIIFF (.tif) file. Scanners and imagers are provided by a number of commercial vendors including Virtek, Packard BioScience, Axon, Applied Precision and others.  Save the fluorescent image as a TIFF (.tif) file. Consult the user manual prior to using any microarray detection instrument.

6. Quantitate data. Open the .tif file and place a quantitation template over the microarray image. Make sure that the template is placed accurately over the spots. Use the “drag tools” to make minor adjustments to template location. Use the quantitation software to acquire the values located inside each template location. Save the results as a comma- or tab-delimited file.  Delimited data can be imported into Spreadsheet and data mining tools and combined with content map information (see below). Quantitation packages are provided with many commercial scanners and imagers, and can also be purchased as separate packages from a number of commercial sources including BioDiscovery, Inc (Marina del Ray, CA).

Table 1 CheckIt Chips Content Map
Click on the links for a complete list of genes, map locations, gene functions, oligonucleotide orientations, organism, putative expression levels, and a GAL file.
CheckIt Chips Content Map.
CheckIt Chips GAL file.

Figure 1. CheckIt™ Chip data using SeeIt™ Universal Probe hybridization. A CheckIt™ Chip was used according to Protocol #2 above. The processed chip was hybridized for 10 min at room temperature (22°C) with 10 µM SeeIt™ Universal Probe, washed according to Protocol #2 and scanned with a ScanArray 3000 (PerkinElmer). The TIFF file was coded to a rainbow palette for ease of viewing. Fluorescent signals are visible at each microarray location as expected. The "marker spots" located in the upper left and lower right corners of each subgrid contain Cy3-labeled control oligonucleotides. Variations in signal intensity are due to differences in 70-mer sequence composition.

Troubleshooting Tips
If sub-optimal results are obtained, please consider the following troubleshooting tips:

High background fluorescence on CheckIt™ Chips.

• Universal probe or labeled probe mixture dried on the microarray surface during the hybridization reaction.  Make sure the sample remains hydrated at all times. Use an ArrayIt®Hybridization Cassette for best results.
• Wash steps were not sufficient.  Longer or more stringent washes may be required when using certain hybridization solutions.

High background fluorescence on microarrays printed “in-house”.

• Impure probe. Use ArrayIt® Fluorescent Probe Purification Kit.
• Insufficient wash conditions. Use ArrayIt® Microarray Wash Buffers 1, 2 & 3.
• Poor coupling chemistry or substrates. Use ArrayIt®SuperClean, SuperAmine and SuperAldehyde Substrates.

No signal on CheckIt™ Chips.

• Microarrays were washed too long or with overly stringent wash buffers or temperatures. ArrayIt® Microarray Wash Buffers A, B & C are recommended.
• Temperature of hybridization too high.

No signal on “in-house” microarrays.

• Poor PCR Amplification or defective cDNA or long oligonucleotide synthesis prior to printing on microarrays.
• Sub-optimal hybridization conditions. SeeIt™ Universal probe should be hybridized at ambient temperature (20-25oC).
• Overly stringent wash conditions (see protocol above).
• Inefficient probe labeling or ribonuclease contamination in RNA.

Recommended Products
Hybridization Cassette or Hybridization Cassette Plus
Microarray High-Speed Centrifuge
High Throughput Wash Station
Super Microarray Slides Substrates
Wash Buffers 1-3