An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.
A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.
Iourov
Ivan Y
IY
National Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe sh.2, 119152 Moscow, Russia.
Soloviev
Ilia V
IV
Vorsanova
Svetlana G
SG
Monakhov
Viktor V
VV
Yurov
Yuri B
YB
D000782:Aneuploidy; D002869:Chromosome Aberrations; D002877:Chromosomes, Human; D002882:Chromosomes, Human, Pair 13; D041321:Chromosomes, Human, X; D015342:DNA Probes; D004314:Down Syndrome / Q000235:genetics; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007223:Infant; D007399:Interphase; D008297:Male; D009030:Mosaicism; D009630:Nondisjunction, Genetic; D010865:Pilot Projects; D015203:Reproducibility of Results; D012984:Software
D015342:DNA Probes