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Malaysia University |
DNA Microarray Hybridization Quality Control Method
Contributor:
Chris Seidel
Overview
cDNAs printed in an array on microscope slides can be visualized by hybridizing the DNA array to random nine-sequence oligonucleotide labeled with Cy. This control verifies that the DNA has printed and adhered to the microscope slide.
Procedure
A. Probe Preparation for Array Hybridization
1. On wet ice in the order listed below, add each of the specified items to 150 pmol of a Cy-labeled 9-mer oligonucleotide DNA probe to the following final concentrations (use the DNA probe prepared in Protocol ID#2216; also see Hint #1):
3X SSC (final concentration)
10 μg of PolyA (final concentration)
0.2% SDS (final concentration, also see Hint #2)
2. Denature the Cy-labeled Oligo Probe at 90°C for 2 min.
3, Centrifuge briefly in a microcentrifuge at maximum speed to bring down any condensation (see Hint #3)
4. Aliquot approximately 20 μl of the Cy-labeled Oligo Probe onto the DNA array.
5. Incubate the slide at room temperature for 3 to 5 min (see Hint #4).
6. Prepare Washing Buffers #1 and #2. Ensure that the wash chambers, slide racks, and vacuum centrifuge are ready and available.
7. Add Washing Buffer #1 and #2 to individual washing chambers (i.e. Coplin Jars).
8. Gently dip the slide rack in and out of Washing Solution #1 several times.
9. Gently dip the slide rack in and out of Washing Solution #2 several times.
10. After the last wash, place the slide rack in the vacuum centrifuge and centrifuge with vacuum for 5 minutes at 500 rpm (see Hint #5).
11. An example of a quality control microarray is shown in Image #1.
Solutions
Wash Solution #2
0.05X SSC
Wash Solution #1
2X SSC
0.2% (w/v) SDS
10% (w/v) SDS
PolyA
1 mg/ml poly-dATP
SSC (20X)
6 M NaCl
pH 7.2
0.6 M Sodium Citrate
BioReagents and Chemicals
SDS
Sodium Citrate
Sodium Chloride
poly-dATP
Protocol Hints
1. The Cy-labeled cDNA Probe can be dried in a vacuum centrifuge to an appropriate volume for adding probe mix components.
2. To avoid precipitation of the SDS, add it last just prior to denaturation. Also, do not be overly concerned with accurately pipetting very small volumes (μl) of SDS.
3. It is important to cool the probe rapidly to room temperature before pipetting it onto the array; however, do not place it on ice because the SDS will precipitate. Some microcentrifuges allow the tube to spin through air (i.e., the back end of the microcentrifuge tube is not protected by a cover). These microcentrifuge types are ideal since the moving air rapidly cools the tube and its contents to room temperature.
4. The Tm for a 9-mer oligonucleotide ranges from approximately 4 to 40°C. The average value will likely be less than room temperature. The major factors to consider in this protocol are incubation temperature, and salt and oligonucleotide concentrations. Optimize each condition empirically for the oligonucleotide probe and DNA array under study.
5. Allow the surface of each slide to dry evenly in order to avoid problems with background signal. It is extremely important that a centrifuge capable of handling a microscope slide rack be used and that it be immediately available after the last wash.
