ARRAY-CGH WORKSHOP PROTOCOL 04/22/2015
Array-based comparative genomic hybridization (aCGH) uses a two-color process to measure DNA copy number changes and copy-neutral Loss of Heterozygosity (LOH) or Uniparental Disomy (UPD)† in an experimental sample relative to a reference (control) sample. The process involves isolating high molecular weight genomic DNA, labeling the DNA with Cyanine- 3 or Cyanine-5 (Cy3 or Cy5) fluorophores, hybridizing labeled target to a DNA microarray, scanning, and data analysis. There are different vendors/protocols available for aCGH, but the general workflow is the same.
DNA MICROARRAY: A fixed surface (typically glass) to which nucleic acids are immobilized. Depending on the application, DNA microarrays may be comprised of cDNA clones, BAC clones, oligonucleotides, PCR products or other materials. This workshop will utilize 60-mer oligonucleotides.
PROBE: The nucleic acid attached to the microarray that is used to bind regions of genomic DNA in the samples. Probes are selected to be unique in the genome based on bioinformatics. Probe density and coverage determine, in part, the resolution of the DNA microarray.
TARGET: This refers to the sample or control genomic DNA after it has been labeled with a fluorescent dye. The target is hybridized with the DNA microarray and binds, by complementary base pairing, with the probes on the array.
† LOH and UPD detection only possible when using microarrays with single nucleotide polymorphism (SNP) probes and a control sample of known genotype.
For Research Use Only. Not for Use in Diagnostic Procedures
STEP 1: RESTRICTION DIGESTION:
The purpose of this step is to fragment the high molecular weight genomic DNA (gDNA) into smaller pieces. Restriction digestion is required for SNP analysis, while other protocols may use other methods, such as sonication, to fragment the DNA. The restriction enzymes Rsa1 and Alu1 (4-base cutters) are used for this reaction. For Agilent CGH+SNP arrays, the corresponding restriction sites are also taken into account for SNP analysis.
1. Thaw 10X Restriction Enzyme Buffer and BSA. Flick the tube to briefly mix and spin in a microcentrifuge. Store on ice. 2. Label four tubes as follows: a. “Control-1” b. “Control-2” c. “Tumor-1” d. “Tumor-2” 3. Add 10 μL of Control DNA (50ng/μL) to the respective control tubes. See the Appendix for notes on DNA sample quality. 4. Add 10 μL of Tumor DNA (50ng/μL) to the respective tumor tubes. 5. Add 10.2 μL of water to each tube, bringing the total volume to 20.2 μL 6. In a separate tube, make a master mix of the following components. Make the master mix on ice in the order indicated below. Mix well by pipetting up and down.
Component mastermix (μL) Nuclease-free water 9.0 10X Restriction Enzyme Buffer 11.7 BSA 0.9 Alu 1 2.25 Rsa 1 2.25 FINAL VOLUME 26.1
7. Add 5.8 µL of the master mix to each of the 4 genomic DNA samples for a total volume of 26 µL. Mix well by pipetting up and down. 8. Transfer the samples to a heat block set to 37°C and incubate for 2 hrs. 9. Transfer the samples to a heat block set to 65°C and incubate for 20 minutes to inactivate the enzymes. Move the sample tubes to ice.
For Research Use Only. Not for Use in Diagnostic Procedures
STEP 2: SAMPLE LABELING:
The purpose of this step is to incorporate fluorescent cyanine dyes into the genomic DNA using the Klenow enzyme and Random priming. Klenow is a DNA polymerase with ‘exo-‘ activity, meaning it doesn’t go back and edit the DNA it creates. Control samples will be labeled with a different dye than tumor samples. The cyanine dyes are incorporated into the DNA strands as they are replicated using Klenow.enzyme.
1. Briefly spin the samples in a centrifuge to remove contents from the tube walls and lids. 2. Add 5 µL of Random Primer to each reaction containing 26 μL of genomic DNA to make a total volume of 31 μL. Use a different tip for each reaction. Mix well by pipetting up and down gently. 3. Transfer samples to a heat block at 95°C and incubate for 3 minutes. 4. Move samples to ice and incubate on ice for 5 minutes. 5. Briefly spin the samples in a centrifuge to remove contents from the tube walls and lids. 6. Add the following components to the “Control-1” and “Control-2” tubes
Component Per Reaction (µL) 5X Reaction Buffer 10.0 10x dNTPs 5.0 Cyanine-3-dUTP 3.0 Exo(-)Klenow 1.0 FINAL VOLUME 19.0
7. Add the following components to the “Tumor-1” and “Tumor-2” tubes
Component Per Reaction (µL) 5X Reaction Buffer 10.0 10x dNTPs 5.0 Cyanine-5-dUTP 3.0 Exo(-)Klenow 1.0 FINAL VOLUME 19.0
8. The total volume of the reactions is now 50 μL. 9. Mix well by gently pipetting up and down. 10. Transfer the samples to a heat block set to 37°C and incubate for 2 hrs. 11. Transfer the samples to a heat block set to 65°C and incubate for 10 minutes to inactivate the enzymes. Move the sample tubes to ice.
For Research Use Only. Not for Use in Diagnostic Procedures
STEP 3: COLUMN PURIFICATION:
The purpose of this step is to remove excess, unincorporated cyanine dye from the reaction, resulting in purified, labeled target DNA.
1. Spin the labeled gDNA samples in a centrifuge for 1 minute to drive the contents off the walls and lid 2. Add 430 μL 1xTE buffer to each reaction tube. 3. Setup four 2-mL collection tubes and place a column in each tube. 4. Load each labeled gDNA onto a separate column. Cover the cap and spin for 10 minutes at 14000 X g. 5. Discard the flow-through and place the column back in the 2-mL collection tube 6. Add 480 μL 1xTE to each column. Spin for 10 minutes at 14000 X g. Discard the flow- through. 7. INVERT the column into a FRESH 2-mL collection tube that has been appropriately labeled. Spin for 1 minute at 1000 X g to collect the purified sample. 8. Add 1xTE to each sample to bring the volume to 41 μL. Mix well by pipetting. 9. Remove 1.5 μL of each sample into a separate tube for specific activity measurement on the NanoDrop or similar instrument. REFER TO THE APPENDIX OF THIS HANDOUT OR THE OFFICIAL AGILENT PROTOCOL FOR INSTRUCTIONS ON SPECIFIC ACTIVITY CALCULATIONS AND YIELDS. 10. Transfer the contents of “Control-1” into “Tumor-1” and mix by pipetting up and down, for a total volume of 79 μL. 11. Transfer the contents of “Control-2” into “Tumor-2” and mix by pipetting up and down, for a total volume of 79 μL.
For Research Use Only. Not for Use in Diagnostic Procedures
STEP 4: HYBRIDIZATION SETUP:
The purpose of this step is to mix the labeled target with the appropriate Cot-1 DNA and buffer for overnight hybridization with the DNA microarray. Cot-1 DNA is used to block non-specific hybridization.
1. To each of the two samples, add the following components:
Component Per Reaction (µL) Cot-1 DNA 12 10x aCGH Blocking Agent 26 2x HI-RPM Hybridization Buffer 130 FINAL VOLUME 168
2. Mix the samples by gently pipetting up and down, then quickly spin in a centrifuge. 3. Transfer samples to a heat block set to 95°C and incubate for 3 min, then immediately transfer sample tubes to a heat block at 37°C for 30 minutes. 4. Remove samples and briefly centrifuge to collect the sample at the bottom of the tube. 5. Add the 181 µL to the gasket slide as demonstrated in class. 6. Put a microarray ‘active side’ down onto the gasket slide as demonstrated in class. 7. Put the chamber together and clamp the assembly tightly by hand. 8. Vertically rotate the assembled chamber to wet the slides and assess the motility of bubbles. 9. Hybridize overnight at 65°C.
NOTE: For Agilent microarrays, the conditions will slightly differ between 1X, 2X, 4X and 8X formats. The protocol provided herein has been for classroom demonstration purposes. The official protocol should be obtained from Agilent for actual microarray hybridizations.
For Research Use Only. Not for Use in Diagnostic Procedures
STEP 5: MICROARRAY WASHING:
The purpose of this step is to remove unbound, labeled target from the glass slide, leaving behind only labeled targets that have hybridized to the corresponding probes by complementary base pairing.
1. Check the liquid level in the microarray to make sure it covers at least half the slide. If too much evaporation has occurred, hybridization to probes in the middle of the array will be diminished. 2. Setup wash staining dishes as demonstrated in class. 3. Follow the times/temperatures of the wash steps based on the table below:
4. Put the hybridization chamber on a flat surface and loosen the thumbscrew. 5. Slide off the clamp assembly and remove the microarray-gasket sandwich, transfer to Dish #1 for disassembly. 6. Without letting go, submerge the ‘sandwich’ into the buffer and pry open the sandwich from the barcode end using forceps. Let the gasket slide drop to the bottom of the staining dish. 7. Remove the microarray slide and place in the 1st wash buffer (Dish #2). After 5 minutes, transfer to the 2nd wash buffer (Dish #3). 8. After 1 minute of Wash Buffer 2 (Dish #3), gently remove the slide from the solution so that it air dries upon removal. The slow removal from the buffer should take about 10 seconds. 9. It is now ready to be scanned and analyzed.
For Research Use Only. Not for Use in Diagnostic Procedures
APPENDIX
DNA Quality:
Accurate assessment of genomic DNA (gDNA) quantity and quality are crucial to the success of aCGH experiments. High quality gDNA should be free of contaminants such as carbohydrates, proteins, and traces of organic solvents. It should also be intact with minimal degradation. It is recommended to measure gDNA concentration with both a spectrophotometer (ex. NanoDrop) and a fluorometer (ex. Qubit) to provide two independent methods of measurement. Ideally the concentration should be at least 50 ng/µl. When using a spectrophotometer, record the A260/A280 and A260/A230 ratios. High-quality gDNA samples have an A260/A280 ratio of 1.8- 2.0, which indicates the absence of contaminating proteins. The ideal 260/230 ratio for pure DNA is >2, which indicates the absence of other contaminants. Gel electrophoresis (ex. slab or TapeStation) is recommended to assess DNA degradation which would appear as a smear of lower molecular weight DNA fragments.
Yield following DNA isolation may be calculated on a spectrophotometer using the formula below:
Degraded gDNA from formalin-fixed, paraffin-embedded (FFPE) tissue may be used for aCGH, however, a different labeling methodology is typically recommended.
Target Yield and Specific Activity Calculations:
For Research Use Only. Not for Use in Diagnostic Procedures
The table below indicates the expected yield and specific activity after labeling and clean-up.
For Research Use Only. Not for Use in Diagnostic Procedures