WHO classification: RAEB I, bm blasts (%): 5, sex/age at diagnosis (years): male/72, karyotype: 46,XY,del(5)(q22q33)[19]/46,XY[2], % del5q (FISH): 85, % del5q (karyogram)86
Extracted molecule
genomic DNA
Extraction protocol
For DNA isolation archieved bone marrow cells were washed in PBS and DNA was isolated with QIAmp DNA Blood Midi Kit (Qiagen, Hilden, Germany) according to the protocol of the manufacturer.
Label
Cy3
Label protocol
Labelling: 1.) Digestion of genomic DNA: Mix the following components on ice in the order stated: 15µl Nuclease free water, 5µl 10x Buffer C (Promega), 2.5µl Alu I (10u/ul)( Promega P/N R6281), 2.5µl Rsa I (10U/ul) (Promega P/N R6371). Dilute 2-5 ug genomic DNA in 25 ul Water and add. Incubate the tubes in a circulating water bath or Thermocycler at 37oC for 2 hours, then transfer on ice. After the reaction is finished bring the volume of the reaction to 100 ul with water. 2.) Purification (Qiagen: QIAprep Miniprep Kit): 1. To prepare Buffer PE: Add ethanol (100%) to buffer PE (supplied with QIAprep Miniprep Kit) (see bottle label for volume). Mark appropriate checkbox to indicate that ethanol was added to bottle. 2. Add 500 µL of Buffer PB (supplied with QIAprep Miniprep Kit) to each 100 ul sample. 3. Apply sample to QIAprep Miniprep column. Centrifuge 60 s at 17,900 × g (13,000 rpm in Eppendorf 5417R) in a microcentrifuge. Discard flow-through. 4. Add 750-µL Buffer PE to each spin column. Centrifuge 60 s at 17,900 × g in a microcentrifuge. Discard flow-through. 5. Centrifuge 60 s at 17,900 × g in a microcentrifuge again. 6. Place the QIAprep spin column in a clean 1.5-mL microfuge tube. To elute DNA, add 25 µL of Buffer EB (10-mM Tris-Cl, pH 8.5) to the center of each spin column. Let stand at room temperature (RT) for 60 s. Centrifuge 60 s at 17,900 × g in a microcentrifuge to collect purified DNA. 7. Samples may be stored at –20 °C prior to labeling. 8. DNA concentration measurements using the NanoDrop ND-1000 UV-VIS spectrophotometer. 3.) Genomic DNA Labeling 1.Calculate volume (µL) of digested genomic DNA for a final 1.5 µg, 2. Transfer volume of digested genomic DNA calculated in step 1 to a nuclease-free 0.5-mL microfuge tube. 3. Add nuclease-free water to bring sample volume to a final volume of 21 µL. 4. Dispense 20-µL aliquots of 2.5× Random Primers Solution (supplied with BioPrime Kit) into each reaction tube. 5. Transfer sample tubes to thermocycler at 95 °C. Incubate at 95 °C for 5.0 min, then move to ice and incubate on ice 5.0 min. Mix the following components on ice: 5.0µl 10x dUTP Mix, 3µl Cy3-dUTP 1.0 mM or Cy3-dUTP 1.0 mM, 1µl ExoKlenow, dispense this 9µl mix into each reaction tube, transfer sample tubes to thermocycler at 37 °C. Incubate at 37 °C for 2 h, remove sample tubes from water bath, add 5.0 µL of Stop Buffer (supplied with BioPrime Kit) to each reaction tube. 4.) Cleanup of Labeled DNA: 1. Combine appropriate Cy5-labeled sample and Cy3-labeled sample for a mixture volume of approximately 100 µL, 2. Add 400 µL of 1× TE (pH 8.0) to each reaction tube, 3. Place Microcon YM-30 filters in a 1.5-mL microfuge tube, and load each sample into filter, 4. Centrifuge 10 min at 8,000 × g (8,700 rpm in Eppendorf 5417R) in a microcentrifuge at room temperature, 5. Discard flow-through, 6. Add 500-µL 1× TE (pH 8.0) to each filter. Centrifuge 10 min at 8,000 × g in a microcentrifuge at room temperature. 7. Discard flow-through. 8. Invert the filter into a fresh 1.5-mL microfuge tube. 9. Centrifuge 1.0 min at 8,000 × g in microcentrifuge at room temperature to collect purified sample. 10. Measure volume (µL) of each eluate until each sample volume is <150 µL. 11. Bring total sample volume to 150 µL with nuclease-free water.
For DNA isolation archieved bone marrow cells were washed in PBS and DNA was isolated with QIAmp DNA Blood Midi Kit (Qiagen, Hilden, Germany) according to the protocol of the manufacturer.
Label
Cy5
Label protocol
Labelling: 1.) Digestion of genomic DNA: Mix the following components on ice in the order stated: 15µl Nuclease free water, 5µl 10x Buffer C (Promega), 2.5µl Alu I (10u/ul)( Promega P/N R6281), 2.5µl Rsa I (10U/ul) (Promega P/N R6371). Dilute 2-5 ug genomic DNA in 25 ul Water and add. Incubate the tubes in a circulating water bath or Thermocycler at 37oC for 2 hours, then transfer on ice. After the reaction is finished bring the volume of the reaction to 100 ul with water. 2.) Purification (Qiagen: QIAprep Miniprep Kit): 1. To prepare Buffer PE: Add ethanol (100%) to buffer PE (supplied with QIAprep Miniprep Kit) (see bottle label for volume). Mark appropriate checkbox to indicate that ethanol was added to bottle. 2. Add 500 µL of Buffer PB (supplied with QIAprep Miniprep Kit) to each 100 ul sample. 3. Apply sample to QIAprep Miniprep column. Centrifuge 60 s at 17,900 × g (13,000 rpm in Eppendorf 5417R) in a microcentrifuge. Discard flow-through. 4. Add 750-µL Buffer PE to each spin column. Centrifuge 60 s at 17,900 × g in a microcentrifuge. Discard flow-through. 5. Centrifuge 60 s at 17,900 × g in a microcentrifuge again. 6. Place the QIAprep spin column in a clean 1.5-mL microfuge tube. To elute DNA, add 25 µL of Buffer EB (10-mM Tris-Cl, pH 8.5) to the center of each spin column. Let stand at room temperature (RT) for 60 s. Centrifuge 60 s at 17,900 × g in a microcentrifuge to collect purified DNA. 7. Samples may be stored at –20 °C prior to labeling. 8. DNA concentration measurements using the NanoDrop ND-1000 UV-VIS spectrophotometer. 3.) Genomic DNA Labeling 1.Calculate volume (µL) of digested genomic DNA for a final 1.5 µg, 2. Transfer volume of digested genomic DNA calculated in step 1 to a nuclease-free 0.5-mL microfuge tube. 3. Add nuclease-free water to bring sample volume to a final volume of 21 µL. 4. Dispense 20-µL aliquots of 2.5× Random Primers Solution (supplied with BioPrime Kit) into each reaction tube. 5. Transfer sample tubes to thermocycler at 95 °C. Incubate at 95 °C for 5.0 min, then move to ice and incubate on ice 5.0 min. Mix the following components on ice: 5.0µl 10x dUTP Mix, 3µl Cy3-dUTP 1.0 mM or Cy3-dUTP 1.0 mM, 1µl ExoKlenow, dispense this 9µl mix into each reaction tube, transfer sample tubes to thermocycler at 37 °C. Incubate at 37 °C for 2 h, remove sample tubes from water bath, add 5.0 µL of Stop Buffer (supplied with BioPrime Kit) to each reaction tube. 4.) Cleanup of Labeled DNA: 1. Combine appropriate Cy5-labeled sample and Cy3-labeled sample for a mixture volume of approximately 100 µL, 2. Add 400 µL of 1× TE (pH 8.0) to each reaction tube, 3. Place Microcon YM-30 filters in a 1.5-mL microfuge tube, and load each sample into filter, 4. Centrifuge 10 min at 8,000 × g (8,700 rpm in Eppendorf 5417R) in a microcentrifuge at room temperature, 5. Discard flow-through, 6. Add 500-µL 1× TE (pH 8.0) to each filter. Centrifuge 10 min at 8,000 × g in a microcentrifuge at room temperature. 7. Discard flow-through. 8. Invert the filter into a fresh 1.5-mL microfuge tube. 9. Centrifuge 1.0 min at 8,000 × g in microcentrifuge at room temperature to collect purified sample. 10. Measure volume (µL) of each eluate until each sample volume is <150 µL. 11. Bring total sample volume to 150 µL with nuclease-free water.
Hybridization protocol
Hybridization: 1. Add the following components in the order indicated to reaction tubes containing 150 l of purified Cy5- and Cy3-labeled sample mixture: 50µl Cot-1 DNA (1.0 mg/ml) (Invitrogen 15279-011), 50µl Agilent 10x Blocking Reagent, 250µl Agilent 2x Hybridization Buffer. 2. Transfer sample tubes to circulating water bath at 95°C. Incubate at 95°C for 3.0 min, 3. then move to ice. 4. Transfer sample tubes to circulating water bath (bzw. Heizblock) 1at 37°C. Incubate at 37°C for 30 min. 5. Remove samples from water bath. 6. Centrifuge 5 min at 17,900 x g (13,000 rpm in Eppendorf 5417R) in microfuge to pellet any precipitates. 7. Carefully remove supernatant materials. Detailed instructions on the use of Surehyb chambers can be found at www.agilent.com. Load a clean SureHyb gasket slide into the chamber base with the label facing up and aligned with the rectangular section of the chamber base. Ensure that the gasket slide is flush with the chamber base and is not ajar. Slowly dispense 490 l of hybridization mixture onto the gasket well in a “drag and dispense” manner. Place an array active side down onto the Surehyb gasket, so the numeric barcode side is facing up. Assess that the sandwich-pair is properly aligned. Place the chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces. Hand-tighten the clamp onto the chamber. Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles. Tap the assembly on a hard surface if necessary to move stationary bubbles. Set your hybridization rotator to rotate at 20 rpm. Hybridize at 65oC for 40 hr. Wash: 1. Completely fill a first slide staining dish with Wash 1 (0.5x SSPE, 0.005% N-lauroylsarcosine) at room temperature. 2. Place slide rack into a second slide staining dish. Add magnetic stir bar. Fill the slide staining dish with enough Wash 1 (0.5x SSPE, 0.005% N-lauroylsarcosine) at room temperature to cover the slide rack. Place this dish on a magnetic stir plate. 3. Remove one hybridization chamber from incubator. 4. Disassemble the hybridization chamber and place the array-gasket sandwich in the first slide staining dish filled with Wash 1 (0.5x SSPE, 0.005% N-lauroylsarcosine) at room temperature. 5. With the sandwich completely submerged in Wash 1 (0.5x SSPE, 0.005% N-lauroylsarcosine), pry the sandwich open with one of the blunt ends of the tweezers. 6. Remove slide and place into slide rack in the second slide staining dish containing Wash 1 (0.5x SSPE, 0.005% N-lauroylsarcosine) at room temperature. Minimize exposure of the slide to air. 7. Repeat steps 7.9.6 and 7.9.7 for all remaining hybridization chambers in group. A maximum of 4 disassembly procedures yielding 4 slides is advised if you are using a 10-slide rack. 8. When all slides in the group are placed into slide rack in staining dish, stir using setting 4 for 5.0 min. 9. During Wash 1 incubation put pre-warmed (37°C) 1.5 liter dish filled with water and containing a slide staining dish on the magnetic stirrer with heating element (setting 3.5). Fill the slide staining dish approximately ¾ full with Wash 2 (0.1x SSPE, 0.005% N-lauroylsarcosine) (pre-warmed to 37°C). Add magnetic stir bar. Maintain temperature of Wash 2 at 37°C. 10. Transfer slide rack to slide staining dish filled with Wash 2 at 37°C, and stir using setting 4 for 1.0 min. 11. Slowly remove slides from Wash2 trying to minimize droplets on the slide. It should take 5 to 10 s. to remove the slide rack. 12. Scan slides slides immediately to minimize impact of environmental oxidants on signal intensities.
Scan protocol
Scanned on Agilent Array scanner G2505B following manufacturers recommendations, scan resolution: 10µm. Images were quantified using Feature Extraction Software (version A.7.5.1, June 2004) (Agilent Technologies).
Description
MDC vs control genomic DNA
Data processing
Linear&LOWESS normalized log10 ratios (red/green) (standard settings of Feature Extraction Software), converted to log2 ratios (test/reference) afterwards
