CGH Protocol_生物行

Thursday, January 29, 2004

Description
CGH is a molecular cytogenetic method of screening a tumor for genetic changes. The alterations are classified as DNA gains and losses and reveal a characteristic pattern that includes mutations at chromosomal and subchromosomal levels.

Procedure
Freeze tissue block (0.5 - 1 cm2) on the cryostat plate, e.g. by applying few drops of isotonic NaCl or water and placing the tissue immediately on the cooled cryostat plate
Cut the tissue block and place first section (5-8 m) on labeled glass slide (close to case No. label)
Cut about 20-30 section (20-30m) and transfer them in the labeled eppendorf tube which contains 900l digestion buffer. The transfer is facilitated by picking the cool sections by the tip of the pasteur pipette. Change pipette after each case!
Transfer last section (5-8 m) on the glass slide (distal to case No. label)
Add 30-50 l of Proteinase K (stock solution of 20 mg/ml)
Incubate for at least 2 hours at 50C, check the digestion by the disintegration if the tissue, add new Proteinase K if the digestion is bad. The Proteinase K digestion can be extended over night or even for several days.
Add 1000 l of Phenol/Chloroform/Isoamylalcohol, mix e.g. by inverting for about 10 min, centrifuge in a table top centrifuge for 10-20 min until the two phases have clearly separated. Discard upper phase.
Repeat step 7 at least once (better: twice).
Add 1/10 vol (about 90 l) 3 M NaCl, mix. Add 1 vol (about 1000 l) ice cool isopropanol. Mix by inverting (white DNA pellet visible? If yes, DNA amount and quality usually oK for Nick translation and CGH)
Centrifuge DNA pellet for about 5 min, discard supernatant, wash once with 100% EtOH and once with 70% EtOH. Finally air dry pellet or dry it by a SpeedVac (5-10 min). If you use a SpeedVac take care not to dry the DNA too much since high molecular, ultradry DNA may be difficult to dissolve
Dissolve pellet in about 100-200l H20 (e.g. Acqua ad iniectabile, Braun Melsungen) depending on the amount of DNA
Determine DNA concentration by a photometer (Final concentration for nick translation should by higher than 150 g/ml).
Check amount of tumor DNA in the tissue block by an H&E stain of the slide (Amount of tumor DNA compared to normal stromal tissue should exceed 70%)

Recipes
Cool cryostat down to -20 to -30C about 3 hours prior to dissection
Label eppendorf tube (2 ml, e.g. Safe Lock) and microscopic slides with the case number
Digestion buffer (50 ml Tris, pH 8.5, 1 mM EDTA, 0.5% Tween 20)
Proteinase K (500 l aliquots of a 20mg/ml stock solution, keep aliquots at -20C)
Melt the tip of long Pasteur pipettes (one pipette per case)
Dissect only one block per time in the cryostat
Keep other tissue block frozen (e.g. in styropor box with liquid nitrogen or dry ice) during dissection
Take care to replace tissue block in the correct vial after dissection

Supplies

Tips

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CGH Protocol