15 Preparation of Samples for CGH by DOP-PCR from microdissected cells
195
5. Centrifuge at 14,000 rpm and 4°C for 30 min.
6. Remove supernatant and wash pellet with 70% ethanol.
7. Spin down again at 14000 rpm and 4°C for 15 min.
8. Remove supernatant and dry pellet.
9. Dissolve pellet in 10 J.lI hybridization mixture.
10. Hybridize the probe cocktail to normal human metaphases according
to standard hybridization protocols for CGH, using an 18x 18 mm coverslip.
Troubleshooting
Problem
PCR product too weak in control gel
Strong PCR product in blank and!or
sham control
No PCR product at all
High fluorescent background in
hybridization
Weak hybridization signal
Reduced homogeneity of hybridization!
granulous signals
References
Approach
increase number of PCR cycles
discard and renew all reagents
use a new batch of Taq-Polymerase
purify the PCR-product before hybridization using e.g. spin column
amplify signals with secondary antibodies,
increase probe concentration
elongate hybridization time (up to 3 days),
modify denaturation temperature and time
and the pretreatment of human metaphases (e.g. pepsin digestion)
Aubele M, Mattis A, Zitzelsberger H, Walch A, Kremer M, Hutzler P, Hofler H, Werner
M (1999) Intratumoral heterogeneity in breast carcinoma revealed by lasermicrodissection and comparative genomic hybridization. Cancer Genet Cytogenet
110:94-102
Isola J, DeVries S, Chu L, Ghazvini S, Waldman F (1994) Analysis of changes in DNA
sequence copy number by comparative genomic hybridization in archival paraffinembedded tumor samples. Am J Pathol 145:1301-8
Jung V, Romeike BFM, Henn W, Feiden W, Moringlane JR, Zang KD, Urbschat S (1999)
Evidence of focal genetic heterogeneity in glioblastoma multiformed by area specific
CGH on microdissected tumor cells. J Neuropath Exp Neurol 58:993-999
195
5. Centrifuge at 14,000 rpm and 4°C for 30 min.
6. Remove supernatant and wash pellet with 70% ethanol.
7. Spin down again at 14000 rpm and 4°C for 15 min.
8. Remove supernatant and dry pellet.
9. Dissolve pellet in 10 J.lI hybridization mixture.
10. Hybridize the probe cocktail to normal human metaphases according
to standard hybridization protocols for CGH, using an 18x 18 mm coverslip.
Troubleshooting
Problem
PCR product too weak in control gel
Strong PCR product in blank and!or
sham control
No PCR product at all
High fluorescent background in
hybridization
Weak hybridization signal
Reduced homogeneity of hybridization!
granulous signals
References
Approach
increase number of PCR cycles
discard and renew all reagents
use a new batch of Taq-Polymerase
purify the PCR-product before hybridization using e.g. spin column
amplify signals with secondary antibodies,
increase probe concentration
elongate hybridization time (up to 3 days),
modify denaturation temperature and time
and the pretreatment of human metaphases (e.g. pepsin digestion)
Aubele M, Mattis A, Zitzelsberger H, Walch A, Kremer M, Hutzler P, Hofler H, Werner
M (1999) Intratumoral heterogeneity in breast carcinoma revealed by lasermicrodissection and comparative genomic hybridization. Cancer Genet Cytogenet
110:94-102
Isola J, DeVries S, Chu L, Ghazvini S, Waldman F (1994) Analysis of changes in DNA
sequence copy number by comparative genomic hybridization in archival paraffinembedded tumor samples. Am J Pathol 145:1301-8
Jung V, Romeike BFM, Henn W, Feiden W, Moringlane JR, Zang KD, Urbschat S (1999)
Evidence of focal genetic heterogeneity in glioblastoma multiformed by area specific
CGH on microdissected tumor cells. J Neuropath Exp Neurol 58:993-999