Affymetrix Microarray Target Hybridization

AFFYMETRIX MICROARRAY TARGET HYBRIDIZATION

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

Invitrogen Life Technologies, P/N 15561-020

3. Herring Sperm DNA:

Promega Corporation, P/N D1811

4. GeneChip Eukaryotic Hybridization Control Kit:

Affymetrix, P/N 900454 (30 reactions) or P/N 900457 (150 reactions), contains Control cRNA and Control Oligo B2

5. Control Oligo B2:

3 nM, Affymetrix, P/N 900301 (can be ordered separately)

6. 5M NaCl:

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

7. MES hydrate SigmaUltra:

Sigma-Aldrich, P/N M5287

8. MES Sodium Salt:

Sigma-Aldrich, P/N M5057

9. EDTA Disodium Salt:

0.5M solution (100 mL), Sigma-Aldrich, P/N E7889

10. DMSO:

Sigma-Aldrich, P/N D5879

11. 10% Surfact-Amps 20 (Tween-20):

Pierce Chemical, P/N 28320

12. 12X MES Stock Buffer (1.22M MES, 0.89M [Na+]) (1 L):

Mix 64.61g of MES hydrate, 193.3g of MES Sodium Salt, 800 mL of Molecular Biology Grade water, Mix and adjust volume to 1 L. The pH should be between 6.5 and 6.7. Filter through a 0.2 μm filte. Do not autoclave. Store at 2°C to 8°C, and shield from light. Discard solution if yellow.

2X Hybridization Buffer (Final 1X concentration is 100 mM MES, 1M [Na+], 20 mM EDTA, 0.01% Tween-20) (50 mL):

Mix 8.3 mL of 12X MES Stock Buffer, 17.7 mL of 5M NaCl, 4.0 mL of 0.5M EDTA, 0.1 mL of 10% Tween-20, 19.9 mL of water. Store at 2°C to 8°C, and shield from light

PROCEDURE - Eukaryotic Target Hybridization

Please refer to the table below for the necessary amount of cRNA required for appropriate probe array format. These recipes take into account that it is necessary to make extra hybridization cocktail due to a small loss of volume (10-20 μL) during each hybridization

1. Mix the following for each target, scaling up volumes for hybridization to multiple probe arrays

NOTE: If using the GeneChip IVT Labeling Kit to prepare the target, a final concentration of 10% DMSO needs to be added in the hybridization cocktail for optimal results

1.1 Hybridization cocktail for single probe array:

Component	49/64 Format	100 Format	169/400 Format	Final
Fragmented cRNA	15 mg	10 mg	5 mg	0.05 mg/mL
Control Oligonucleotide B2 (3 nM)	5 mL	3.3 mL	1.7 mL	50 pM
20X Eukaryotic Hybridization Controls (bioB, bioC, bioD, cre)	15 mL	10 mL	5 mL	1.5, 5, 25, and 100 pM respectively
Herring Sperm DNA (10 mg/mL)	3 mL	2 mL	1 mL	0.1 mg/mL
BSA (50 mg/mL)	3 mL	2 mL	1 mL	0.5 mg/mL
2X Hybridization buffer	150 mL	100 mL	50 mL	1X
DMSO	30 mL	20 mL	10 mL	10%
H₂O	to 300 mL	to 200 mL	to 100 mL
Final volume	300 mL	200 mL	100 mL

NOTE: It is imperative that frozen stocks of 20X GeneChip Eukaryotic Hybridization Controls are heated to 65°C for 5 minutes to completely resuspend the cRNA before aliquotting

2. Equilibrate probe array to room temperature immediately before use

NOTE: It is important to allow the arrays to equilibrate to room temperature completely. Specifically, if the rubber septa are not equilibrated to room temperature, they may be prone to cracking, which can lead to leaks

3. Heat the hybridization cocktail to 99°C for 5 minutes in a heat block

4. Meanwhile, wet the array by filling it through one of the septa with appropriate volume of 1X Hybridization Buffer using a micropipettor and appropriate tips:

Array	Hybridization Volume	Total Fill Volume
49 Format (Standard)	200 μL	250 μL
64 Format	200 μL	250 mL
100 Format (Midi)	130 μL	160 mL
169 Format (Mini)	80 mL	100 mL
400 Format (Micro)	80mL	100 mL

NOTE: It is necessary to use two pipette tips when filling the probe array cartridge: one for filling and the second to allow venting of air from the hybridization chamber

5. Incubate the probe array filled with 1X Hybridization Buffer at 45°C for 10 minutes with rotation

6. Transfer the hybridization cocktail that has been heated at 99°C, in step 3, to a 45°C heat block for 5 minutes

7. Spin hybridization cocktail(s) at maximum speed in a microcentrifuge for 5minutes to remove any insoluble material from the hybridization mixture

8. Remove the buffer solution from the probe array cartridge and fill with appropriate volume of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube.

9. Place probe array into the Hybridization Oven, set to 45°C. Avoid stress to the motor; load probe arrays in a balanced configuration around the
axis. Rotate at 60 rpm

10. Hybridize for 16 hours

NOTE: During the latter part of the 16-hour hybridization, prepare reagents required immediately after completion of hybridization

To wash, stain, and scan the microarray, go to protocol Washing, Staining, and Scanning Affymetrix Microarrays (152.html)