Washing, Staining, and Scanning Affymetrix Microarrays

WASHING, STAINING, and SCANNING AFFYMETRIX MICROARRAYS

Last Update: December 2006

Based on the Affymetrix Technical Manual GeneChip Expression Analysis

PREPARE SOLUTIONS

1. Water, Molecular Biology Grade:

BioWhittaker Molecular Applications/ Cambrex, P/N 51200

2. Distilled water:

Invitrogen Life Technologies, P/N 15230-147

3. Bovine Serum Albumin (BSA) solution (50 mg/mL):

Invitrogen Life Technologies, P/N 15561-020

4. R-Phycoerythrin Streptavidin:

Molecular Probes, P/N S-866

5. 5M NaCl:

RNase-free, DNase-free, Ambion, P/N 9760G

6. PBS, pH 7.2:

Invitrogen Life Technologies, P/N 20012-027

7. 20X SSPE:

(3M NaCl, 0.2M NaH₂PO₄, 0.02M EDTA), BioWhittaker Molecular Applications/Cambrex, P/N 51214

8. Goat IgG, Reagent Grade:

Sigma-Aldrich, P/N I 5256

9. Anti-streptavidin antibody (goat), biotinylated:

Vector Laboratories, P/N BA-0500

10. 10% Surfact-Amps 20 (Tween-20):

Pierce Chemical, P/N 28320

11. Wash Buffer A: Non-Stringent Wash Buffer (6X SSPE, 0.01% Tween-20) (1 L):

Mix 300 mL of 20X SSPE, 1.0 mL of 10% Tween-20, and 699 mL of water. Filter through a 0.2 μm filter

12. Wash Buffer B: Stringent Wash Buffer (100 mM MES, 0.1M [Na+], 0.01% Tween-20) (1 L):

Mix 83.3 mL of 12X MES Stock Buffer, 5.2 mL of 5M NaCl, 1.0 mL of 10% Tween-20, and 910.5 mL of water. Filter through a 0.2 μm filter and store at 2°C to 8°C and shield from light

13. 2X Stain Buffer (Final 1X concentration: 100 mM MES, 1M [Na+], 0.05% Tween-20) (250 mL):

Mix 41.7 mL of 12X MES Stock Buffer, 92.5 mL of 5M NaCl, 2.5 mL of 10% Tween-20 and 113.3 mL of water. Filter through a 0.2 μm filter and store at 2°C to 8°C and shield from light

14. 10 mg/mL Goat IgG Stock:

Resuspend 50 mg in 5 mL of 150 mM NaCl. Store at 4°C

Experiment and Fluidics Station Setup

PROCEDURE - Step 1 - Defining File Locations

1. Launch Microarray Suite from the workstation and select Tools→Defaults→File Locations from the menu bar.

2. The File Locations window displays the locations of the following files:

2.1 Probe Information (library files, mask files)

2.2 Fluidics Protocols (fluidics station scripts)

2.3 Experiment Data (.exp, .dat, .cel, and .chp files are all saved to location selected here)

3. Verify that all three file locations are set correctly and click OK

PROCEDURE - Step 1 - Entering Experiment Information

NOTE: To wash, stain, and scan a probe array, an experiment must first be registered in GCOS or Microarray Suite. Please follow the instructions detailed in the “Setting Up an Experiment” section of the appropriate GCOS or Microarray Suite User’s Guide

1. The fields of information required for registering experiments in Microarray Suite are:

1.1 Experiment Name

1.2 Probe Array Type

2. In GCOS, three additional fields are required:

2.1 Sample Name

2.2 Sample Type

2.3 Project

PROCEDURE - Step 1 - Preparing the Fluidics Station

The Fluidics Station 400, or 450/250 is used to wash and stain the probe arrays. It is operated using GCOS/Microarray Suite

Setting Up the Fluidics Station:

1. Turn on the Fluidics Station using the toggle switch on the lower left side of the machine

2. Select Run → Fluidics from the menu bar

The Fluidics Station dialog box appears with a drop-down list for selecting the experiment name for each of the fluidics station modules. A second drop-down list is accessed for choosing the Protocol for each of the fluidics station modules

NOTE: Refer to the Fluidics Station User’s Guide for instructions on connecting and addressing multiple fluidics stations

Priming the Fluidics Station:

Priming ensures that the lines of the fluidics station are filled with the appropriate buffers and the fluidics station is ready for running fluidics station protocols

Priming should be done:

¡ when the fluidics station is first started

¡ when wash solutions are changed

¡ before washing, if a shutdown has been performed

¡ if the LCD window instructs the user to prime

1. To prime the fluidics station, select Protocol in the Fluidics Station dialog box

2. Choose Prime or Prime_450 for the respective modules in the Protocol drop-down list

3. Change the intake buffer reservoir A to Non-Stringent Wash Buffer, and intake buffer reservoir B to Stringent Wash Buffer

4. For MAS, click Run for each module to begin priming. In GCOS, select the All Modules check box, then click Run

Probe Array Wash and Stain

After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of Non-Stringent Wash Buffer (Wash Buffer A)

NOTE: If necessary, at this point, the probe array can be stored at 4°C for up to 3 hours before proceeding with washing and staining. Equilibrate the probe array to room temperature before washing and staining

This protocol is recommended for use with probe arrays with probe cells of 24 μm or smaller. This procedure takes approximately 90 minutes to complete

Preparing the Staining Reagents:

Prepare the following reagents. Volumes given are sufficient for one probe array

SAPE Stain Solution:

Streptavidin Phycoerythrin (SAPE) should be stored in the dark at 4°C, either foil-wrapped or kept in an amber tube. Remove SAPE from the refrigerator and tap the tube to mix well before preparing stain solution. Do not freeze SAPE. Always prepare the SAPE stain solution fresh, on the day of use

Prepare SAPE Solution Mix: Mix 600 mL of 2X Stain Buffer, 48 mL of 50 mg/mL BSA, 12 mL of 1 mg/mL Streptavidin Phycoerythrin (SAPE), and 540 mL of distilled water for a total volume of 1,200 mL

Mix well and divide into two aliquots of 600 μL each to be used for stains 1 and 3

Prepare Antibody Solution Mix: Mix 300 mL of 2X Stain Buffer, 24 mL of 50 mg/mL BSA, 6 mL of 10 mg/mL Goat IgG Stock, 3.6 mL of 0.5 mg/mL biotinylated antibody, and 266.4 mL of distilled water for a total volume of 600 mL

PROCEDURE - Washing and Staining the Probe Array using a FS-450

1. In the Fluidics Station dialog box on the workstation, select the correct experiment name from the drop-down Experiment list

⇒The Probe Array Type appears automatically

2. In the Protocol drop-down list, select the appropriate antibody amplification protocol to control the washing and staining of the probe array format being used

3. Choose Run in the Fluidics Station dialog box to begin the washing and staining. Follow the instructions in the LCD window on the fluidics station

4. Insert the appropriate probe array into the designated module of the fluidics station while the cartridge lever is in the down, or eject, position. When finished, verify that the cartridge lever is returned to the up, or engaged, position

5. Remove any microcentrifuge vial remaining in the sample holder of the fluidics station module(s) being used

6. If prompted to “Load Vials 1-2-3,” place the three experiment sample vials (the microcentrifuge vials) into the sample holders 1, 2, and 3 on the fluidics station

6.1 Place one vial containing 600 μL of streptavidin phycoerythrin (SAPE) solution in sample holder 1

6.2 Place one vial containing 600 μL of anti-streptavidin biotinylated antibody solution in sample holder 2

6.3 Place one vial containing 600 μL of streptavidin phycoerythrin (SAPE) solution in sample holder 3

6.4 Press down on the needle lever to snap needles into position and to start the run

The run begins. The Fluidics Station dialog box at the workstation terminal and the LCD window display the status of the washing and staining as they progress

7. At the end of the run, or at the appropriate prompt, remove the microcentrifuge vials and replace with three empty microcentrifuge vials

8. Remove the probe arrays from the fluidics station modules by first pressing down the cartridge lever to the eject position

9. Check the probe array window for large bubbles or air pockets

NOTE: If you do not scan the arrays right away, keep the probe arrays at 4°C and in the dark until ready for scanning

10. If there are no more samples to hybridize, shut down the fluidics station following the procedure outlined in the section, Shutting Down the Fluidics Station

PROCEDURE - Probe Array Scan

Handling the GeneChip Probe Array

1. On the back of the probe array cartridge, clean excess fluid from around septa

2. Carefully apply one Tough-Spots to each of the two septa. Press to ensure that the spots remain flat. If the Tough-Spots do not apply smoothly, that is, if you observe bumps, bubbles, tears, or curled edges, do not attempt to smooth out the spot. Remove the spot and apply a new spot

3. Insert the cartridge into the scanner and test the autofocus to ensure that the Tough-Spots do not interfere with the focus. If you observe a focus error message, remove the spot and apply a new spot. Ensure that the spots lie flat

Scanning the Probe Array

1. Select Run → Scanner from the menu bar. Alternatively, click the Start Scan icon in the tool bar

⇒The Scanner dialog box appears with a drop-down list of experiments that have not been run

2. Select the experiment name that corresponds to the probe array to be scanned. A previously run experiment can also be selected by using the Include Scanned Experiments option box. After selecting this option, previously scanned experiments appear in the drop-down list

3. By default, for the GeneArray Scanner only, after selecting the experiment the number [2] is displayed in the Number of Scans box to perform the recommended 2X image scan. For the GeneChip® Scanner 3000, only one scan is required

4. Once the experiment has been selected, click the Start button

⇒A dialog box prompts you to load an array into the scanner

5. If you are using the GeneArray® Scanner, click the Options button to check for the correct pixel value and wavelength of the laser beam

5.1 Pixel value = 3 μm

5.2 Wavelength = 570 nm

NOTE: If you are using the GeneChip Scanner 3000, pixel resolution and wavelength are preset and cannot be changed

6. Open the sample door on the scanner and insert the probe array into the holder. Do not force the probe array into the holder. Close the sample door of the scanner

7. Click OK in the Start Scanner dialog box

⇒The scanner begins scanning the probe array and acquiring data. When Scan in Progress is selected from the View menu, the probe array image appears on the screen as the scan progresses

Once the scan is complete, the data is ready for analysis

Shutting Down the Fluidics Station

1. After removing a probe array from the probe array holder, the LCD window displays the message ENGAGE WASHBLOCK

2. If you are using the FS-400, latch the probe array holder by gently pushing up until a light click is heard. Engage the washblock by firmly pushing up on the cartridge lever to the ENGAGE position

If you are using the FS-450, gently lift up the cartridge lever to engage, or close, the washblock

⇒The fluidics station automatically performs a Cleanout procedure. The LCD window indicates the progress of the Cleanout procedure

3. When the fluidics station LCD window indicates REMOVE VIALS, the Cleanout procedure is complete

4. Remove the sample microcentrifuge vial(s) from the sample holder(s)

5. If no other hybridizations are to be performed, place wash lines into a bottle filled with deionized water

6. Choose Shutdown or Shutdown_450 for all modules from the drop-down Protocol list in the Fluidics Station dialog box. Click the Run button for all modules. The Shutdown protocol is critical to instrument reliability. Refer to the appropriate Fluidics Station User’s Guide for more information

7. After Shutdown protocol is complete, flip the ON/OFF switch of the fluidics station to the OFF position

NOTE: To maintain the cleanliness of the fluidics station and obtain the highest quality image and data possible, a weekly bleach protocol and a monthly decontamination protocol are highly recommended