98
MORIMASA MATSUTA AND MAYUMI MATSUTA
Troubleshooting
• Poor hybridization: Poor quality of metaphase slides is inadequate.
• Dim signal and/or Granular hybridizations: Probe is too small
or big. Size range should be about 500-2,000 bp. If the probe
fragments are longer, increase the incubation time or the
amount of enzyme mixture and if shorter, decrease the incubation time or the amount of enzyme mixture. Probe concentration is low. Increase amount of probe.
• Chromosomes cannot be identified: Ensure temperature of
denaturation solution is 74°C.
Acknowledgements
The principal of FISH described in this Manual was originally
developed by Pinkel et al. at Laurence Livermore National
Laboratory, California, USA and that of CGH by Kallioniemi
et al. at University of California, San Francisco, California,
USA. They have been modified at University of California,
San Francisco and Tampera University, Tampera, Finland.
The authors express special thanks to the technical staff at Vysis
Inc. and Fujisawa Ltd. for their helpful advice, especially in Trouble shooting and Codenaturation sessions.
References
Du Manoir S, Kallioniemi OP, Lichter P et al (1995) Hardware and software
requirements for quantative analysis of comparative genomic hybridization. Cytometry 19:4-9
Houldsworth J, Chaganti RSK (1994) Comparative genomic hybridization:
An overview. Am J PatholI45:1253-1260
Kallioniemi A, Kallioniemi OP, Sudar D, Rutovits D, Gray JW, Waldman
FM, Pinkel D (1992) Comparative genomic hybridization for molecular
cytogenetic analysis of solid tumors. Science 258:818-821
Kallioniemi OP, Kallioniemi A, Kurisu W, Thor A, Yu LC, Smith HS, Waldman FM, Pinkel D, Gray JW (1992) ERBB2 amplification in breast cancer
analyzed by fluorescence in situ hybridization. Proc. Natl. Acad. Sci.
USA. 89; 5321-5325
MORIMASA MATSUTA AND MAYUMI MATSUTA
Troubleshooting
• Poor hybridization: Poor quality of metaphase slides is inadequate.
• Dim signal and/or Granular hybridizations: Probe is too small
or big. Size range should be about 500-2,000 bp. If the probe
fragments are longer, increase the incubation time or the
amount of enzyme mixture and if shorter, decrease the incubation time or the amount of enzyme mixture. Probe concentration is low. Increase amount of probe.
• Chromosomes cannot be identified: Ensure temperature of
denaturation solution is 74°C.
Acknowledgements
The principal of FISH described in this Manual was originally
developed by Pinkel et al. at Laurence Livermore National
Laboratory, California, USA and that of CGH by Kallioniemi
et al. at University of California, San Francisco, California,
USA. They have been modified at University of California,
San Francisco and Tampera University, Tampera, Finland.
The authors express special thanks to the technical staff at Vysis
Inc. and Fujisawa Ltd. for their helpful advice, especially in Trouble shooting and Codenaturation sessions.
References
Du Manoir S, Kallioniemi OP, Lichter P et al (1995) Hardware and software
requirements for quantative analysis of comparative genomic hybridization. Cytometry 19:4-9
Houldsworth J, Chaganti RSK (1994) Comparative genomic hybridization:
An overview. Am J PatholI45:1253-1260
Kallioniemi A, Kallioniemi OP, Sudar D, Rutovits D, Gray JW, Waldman
FM, Pinkel D (1992) Comparative genomic hybridization for molecular
cytogenetic analysis of solid tumors. Science 258:818-821
Kallioniemi OP, Kallioniemi A, Kurisu W, Thor A, Yu LC, Smith HS, Waldman FM, Pinkel D, Gray JW (1992) ERBB2 amplification in breast cancer
analyzed by fluorescence in situ hybridization. Proc. Natl. Acad. Sci.
USA. 89; 5321-5325