COMPARATIVE GENOMIC HYBRIDIZATION (CGH) PROTOCOL

For CGH, selected slides with normal metaphase chromosomes are used (stock at -20°C). Prior to the making of the chromosome slides, slides are coated with silane.

SAVING OF DAPI-IMAGES

-DAPI-staining solution : 100 ml 1xPBS + 2 µl DAPI-stocksolution(1mg/ml)
-incubate slides for 3' in the stainingsolution at RT in the dark
-rinse 3x1' with tap water
-air dry slides and mount in antifade medium (35 µl Vectashield/slide)
-15 to 20 mitoses are selected and DAPI images are captured using a black and white CCD camera .

SLIDE PRETREATMENT

Prior to slide pretreatment coverslips are gently removed and slides are rinsed for 15' with 94% ethanol at RT . Subsequently slides are air dried.

1)RNase pretreatment
-incubate slides 1 hour at 37°C in 100 µg/ml RNase A in 2xSSC (pH 7) in moist chamber
Stock RNase A : 10 mg/ml Dilution : 10 µl stock + 990 µl 2xSSC
-wash 3x5' with 2xSSC (RT)
-dehydratation in ethanol series : 70%,90%,94% for 3' ( RT) on shaker
-air dry slides

2)Pepsin treatment
-incubate 30' at 37°C(WWB) in 0.005% pepsin in 0.01 N HCl
Stock pepsin :10% worksolution : 0.005% (50 µl stock + 100 ml HCl both pre-warmed at 37°C)
-wash in 5' 1xPBS (RT)

3)Postfixation
-wash 5' in postfixation buffer at RT on shaker :
100 ml postfixation-buffer : 10 ml 10xPBS, 10 ml 0.5 M MgCl2, 80 ml bidest
-5' fixation in 4% paraformaldehyde (PFA) at RT on shaker
100 ml 4% PFA : 10 ml 10xPBS, 10 ml MgCl2, 30 ml bidest, 50 ml 8% PFA
-wash in 1xPBS 5' at RT on shaker
-dehydratation in ethanol series : 70%,90%,94% for 3' each at RT on shaker
-air dry slides

PROBE PREPARATION (on ice)

1. 300 ng biotinylated test DNA (stock : 20ng/ml) and 300 ng dig-labeled control DNA (stock 20ng/µl) are combined. Add 25´COT-I-DNA (=15 µl of stock-conc.,1 µg/ml)
2. precipitate with Na-acetate (3 M,pH 5.6) : 1/10 of the total volume of the DNA and ice-cold 100% ethanol : 2.5x of the total volume of the DNA
3. chill on ice for 30'
4. centrifuge 30' at 14000 RPM at 4°C
5. remove supernatans (with fine-needle in flow-bench)
6. air dry pellet for 15' (dust free)
7. dissolve the pellet in an appropriate volume of 50% formamide/12.5% dextran- sulphate/2xSSCP (10 µl/slide)
8. incubate 30' at 37°C(WWB) to dissolve the pellet completely
9. denature probes at 70°C(WWB) for 5'
10. chill immediately on ice for 3'
11. preannealing of the probes at 37°C(WWB) for 30'
12. denature target DNA (slides) with 100 µl 70% formamide/2xSSCP for 2.5' under a coverslip on a hot plate at 75°C . The denaturation is started during the last 15' of the preannealing of the probe.
13. chill slides in ice-cold 70% ethanol for 2.5' followed by rinses in 90% and 94% ethanol
14. dry slides on a hot plate (40°C) and apply 10 µl of preannealed probe under coverslip (18x18 mm or 24x24 mm)
15. 3 or 4 days hybridization in moist chamber (60% form./2xSSCP) at 37°C in incubator

PROBE DETECTION

PREPARATION OF SOLUTIONS :
-50% formamide/2xSSC pH 7.0 300 ml :

-150 ml 100% formamide

-30 ml 20xSSC

-120 ml bidest
adjust to pH 7.0 with 6 N HCl
-0.1xSSC
These solutions are both pre-warmed at 42°C
-2xSSC
-4xSSC/Tween-20 0.05% (0.5 ml Tween-20/1l 4xSSC)

1. Washing

-3x5' with 50% formamide/2xSSC at 42°C (WWB)
-5x2' with 0.1xSSC at 42°C
-1x5' with 2xSSC at RT on shaker

2.Blocking

-incubate 20' in moist chamber at RT with 100 µl of 4xSCC/Tween

3. Washing

-1x5' in 4xSSC/Tween (RT)

4. Detection

Layer 1 :

Neutralite-Avidin-FITC

dilution : 1/200 in 4xSSC/Tween
-centrifuge 2' on 12000 RPM
-incubate with 100 µl/slide in moist in dark at 37°C for 30'

Wash 3x5' in 4xSSC/Tween at RT on shaker

Layer 2 :

biotinylated goat-anti-Avidin + mouse anti-dig

dilution : 1/100 + 1/500 in 4xSSC/Tween
-centifuge for 2' on 12000 RPM
-incubate with 100 µl/slide in moist in dark at 37°C for 45'

Wash 3x5' in 4xSSC/Tween at RT on shaker

Layer 3 :

Neutralite-Avidin-FITC + sheep anti-mouse dig

dilution : 1/200 + 1/100 in 4´SCC/Tween
-centifuge for 2' on 12000 RPM
-incubate with 100 µl/slide in moist in dark at 37°C for 45'

Wash 3x5' in 4xSSC/Tween at RT on shaker

Layer 4 :

sheep anti-dig-TRITC

dilution : 1/100 in 4xSSC/Tween
-centifuge for 2' on 12000 RPM
-incubate with 100 µl/slide in moist in dark at 37°C for 45'

Wash 2x5' in 4xSSC/Tween (RT)

5. Counterstaining with DAPI

-incubate for 3' in DAPI-stainingsolution (100 ml 4xSSC/Tween + 2 µl DAPI-stocksolution (1mg/ml)) at RT in the dark
-rinse 3x1'with tap water, air dry and mount in antifade medium (=35 µl Vectashield/slide)

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