title: KIST custom metabolite Agilent 4x44 K array
status: Public on Jul 24 2010
contact.city: Seoul
contact.first: Sailendra
contact.middle: Nath
contact.last: Sarma
contact.email: sarma@kist.re.kr
contact.address: P.O. Box 131, Cheongryang
contact.country: South Korea
contact.institute: Korea Institute of Science and Technology
contact.phone: +8229585080
organism: Homo sapiens
manufacture_protocol: Materials
  MAUI Mixer A4 Hybridization Chambers (Cat No. 02-A008-25)
  A4/LC Hinged Clamp Assembly (Cat No. 02-A004-00)
  SL/A4/LC Assembly/Disassembly Jig (Cat No. 02-033-03)
  Gasket Brayer (Cat No. 02-054-00)
  MAUI Humid Cover (Cat No. 02-D0031-00)
  M-100 Microman® Pipette (Cat No. 01-014-00)
  CP-100 Pipette Tips (Cat No. 01-015-00)
  Port Plugs (Cat No. 03-D0002-00)
  Port Plug Insertion Tool (Cat No. 04-D0005-00)
  Cotton Swabs
  1.5 ml Eppendorf® Tubes
  Hybridization Buffer (see recipe below)
  250 μl Pipette Conditioning Solution (see recipe below- used only to eliminate air from the pipette tip prior to filling the
  hybridization chamber and not for the actual hybridization)
  Additional BioMicro Systems Instructions & Procedures
  Replacing Bay Clamp Assemblies on MAUI Hybridization Systems (CDP-02)
  MAUI Humid Cover for the MAUI Hybridization System (CDP-12)
  MAUI Mixer Sealing Using Port Plugs (CDP-13)
  MAUI Mixer-Slide Assembly Procedure (see MAUI Hybridization System User’s Guide) (CDP-14)
  MAUI Mixer Filling and Sealing Procedure (see MAUI Hybridization System User’s Guide) (CDP-14)
  Modifications to Agilent Two-Color Microarray-Based Gene Expression Analysis --
  Quick Amp Labeling and Hybridization Protocol
  Below is a list of key adaptations made to the Agilent gene expression array labeling (Quick Amp Labeling) and
  hybridization protocol when using the MAUI Hybridization System and 4x44k CGH arrays.
  Table 1: Protocol Differences
  Agilent SureHyb® System MAUI Hybridization System
  Labeled Target(s) - Dry using a SpeedVac®
  Final Sample Volume
  4 x 44K array 110 μl 25 μl
  Hybridization Mixture No formamide Add formamide for a final
  concentration of 16%
  Hybridization Temperature 65ºC 55ºC
  Hybridization Time
  4 x 44K array 17 hours 17 hours
  Total RNA labeling should be performed according to Agilent Quick Amp Labeling protocol. For the microarray
  hybridization process, following removal of the unincorporated dyes it is necessary to reduce the volume of the labeled
  target in order to load it into the ultra-low volume MAUI Mixers. The following modifications have been made to the
  protocol of Agilent Two-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling, version 5.7, March
  2008):
  MAUI® Mixer A4 on Agilent® 4 x 44K Gene Expression Arrays
  Following the purification of the labeled and amplified RNA (step 9, page 24).
  1. Combine the appropriate purified Cy5 and Cy3 labeled RNA samples.
  2. Dry combined samples using an appropriate speed vacuum under low heat and protect samples from light during the
  process (e.g. using aluminum foil). This may take 20 minutes or longer depending on the degree of vacuum, the
  sample volume and total number of samples being prepared.
  3. Prepare the hybridization mixture according to the following recipe at room temperature.
  Note: Make the appropriate volume of hybridization mixture based on the number of samples prepared.
  Table 2: Hybridization Mixture Recipe
  Component Volume
  Nuclease-Free Water 80.6 μl
  Agilent 10X GE Blocking Agent 25 μl
  25x Fragmentation Buffer 4.4 μl
  Final Hybridization Mixture Volume 110 μl
  4. Add 11 μl of the above hybridization mixture to each dried sample in a 1.5 ml Eppendorf Tube. Completely
  resuspend the labeled sample(s) by gently pipetting the hybridization mixture up and down several times (avoid
  causing bubbles). Briefly spin.
  5. Incubate the samples at 60ºC for exactly 30 minutes to fragment RNA.
  6. Add 4 ul of high-quality de-ionized formamide and 10 μl of 2x GE Hybridization Buffer to each sample to stop the
  fragmentation reaction and make a final total volume of 25 μl for each array. Mix well but gently by pipetting. To
  avoid introducing bubbles, do not vortex. Please note that the final concentration of formamide is 16% and the final
  concentration of Agilent 2x GE hybridization buffer in the final hybridization mixture is 0.8x (not 1x), which is
  optimized specifically for the MAUI Hybridization system.
  Final Volume = Chamber Volume + (5 to 10 μl).
  Note: The chamber volumes may vary slightly from lot to lot of MAUI Mixers. The actual chamber volume for each MAUI
  Mixer is indicated on the MAUI Mixer packaging.
  7. Spin tubes (highest speed for 1 minute) to collect the buffer at the bottom of the tubes and load samples as soon as
  possible.
  Protocol
  Prior to using Agilent microarrays with the MAUI Hybridization System you will need to replace the standard bay clamp
  assemblies with A4/LC bay clamp assemblies (see Bay Clamp Replacement Instructions CDP-02).
  1. Set the MAUI Hybridization System temperature to 55ºC.
  2. Prepare 250 μl of Pipette Conditioning Solution in a 1.5 ml Eppendorf tube and warm to 55ºC. The purpose of this
  solution is to eliminate air from the pipette tip prior to aspirating the hybridization mixture for injection into the MAUI
  Mixer hybridization chamber.
  Table 3: Pipette Conditioning Solution Recipe
  Component Volume
  Nuclease-Free Water 80.6 μl
  Agilent 10X Blocking Agent 25 μl
  25x Fragmentation Buffer 4.4 ul
  Agilent 2x GE Hybridization Buffer 100 μl
  High Quality De-Ionized Formamide 40 μl
  Final Pipette Solution Volume 250 μl
  - 3 -
  CDP-20-REVA DEC 2008
  3. Place the solution at 55ºC a few minutes before use.
  4. Open a single MAUI Mixer A4. Attach the mixer to the Agilent microarray slide using an SL/A4/LC
  Assembly/Disassembly jig, as described in the MAUI Hybridization System User’s Guide. Make sure the “active
  array side” of the slide faces up and the barcode end slides into the jig first.
  Note: Completely bray all portions of the adhesive gasket. The adhesive gasket will appear clear (not cloudy) when sealed
  correctly. Do not use excessive pressure when braying directly over the microarray surface, as this may result in damage to
  the array.
  BioMicro Systems highly recommends that you practice assembling and loading MAUI Mixers with used Agilent microarrays
  and Pipette Solution containing 1% Bromophenol Blue.
  5. Fill a single chamber of the MAUI Mixer A4.
  a. Set the M-100 Microman Pipette to 3μl more than the chamber volume indicated on the MAUI Mixer
  package label.
  b. Use a new CP-100 pipette tip and slowly pipette the pipette conditioning solution (step 2) up and down until
  all air bubbles are removed from the tip.
  c. With the pipette plunger depressed, transfer the pipette tip into the tube containing the hybridization
  solution.
  d. Slowly aspirate hybridization solution (being careful not to introduce bubbles into the tip).
  e. Insert the pipette tip into the fill port of the desired chamber. Be sure to hold the pipette tip as vertically as
  possible.
  MAUI Mixer A4
  f. With a continuous motion, slowly inject the hybridization solution into the chamber. After filling, DO NOT
  wipe hybridization solution from fill and vent ports.
  6. Seal the chamber using port plugs (see Port Plug Instructions).
  a. Hold the star shaped port plug carrier to the side of the MAUI Mixer (not over it) with the raised marks on
  the handle facing up.
  b. Center a port plug over the fill port. You should be able to feel the end of the plug insert into the port.
  c. Vertically center the port plug insertion tool over the aligned port plug. Using the tool, lightly (~ 2-4 lbs. of
  pressure) push the port plug into the fill port. Tilt the tool slightly toward the carrier (away from the center of
  the MAUI Mixer) and gently pull the carrier away from the tool. The edge of the port plug tool will cut/snap
  the port plug off of the carrier.
  d. Move the port plug tool back to a vertical position and push with just enough force to fully insert plug. Avoid
  using excessive force, which may disrupt the gasket seal.
  e. Slowly release the pressure on the port plug insertion tool to prevent air from being pulled into the vent port.
  f. Seal the vent port in the same manner (always seal fill port first and then the vent port).
  g. Use a cotton swab to blot excess buffer from around the sealed port plugs until the cotton swab appears
  dry.
  - 4 - CDP-20-REVA DEC 2008
  h. Press lightly on the center