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Chapter 13
Quantitative Fluorescence In Situ Hybridization (QFISH)
Ivan Y. Iourov
Abstract
Fluorescence in situ hybridization (FISH) has a wide spectrum of applications in current molecular cytogenetic and cancer research. This is a unique technique that can be used for chromosomal DNA analy- sis in all cell types, at all stages of the cell cycle, and at molecular resolution. Recent developments in microscopy and imaging systems have allowed quantifi cation of digital FISH images (quantitative FISH or QFISH) and have provided a new way for molecular cytogenetic analysis at single-cell level. QFISH can be applied for studying chromosome imbalances in interphase nuclei or metaphase spreads, measuring rela- tive DNA content at chromosomal loci and identifying parental origin of homologous chromosomes.
Here, a QFISH protocol suitable for the majority of DNA probes using the popular US National Institute of Health developed ImageJ software is described.
Key words Chromosome abnormalities , DNA probes , Fluorescence in situ hybridization , Interphase , Nucleus , Quantifi cation , QFISH
1 Introduction
Quantitative fl uorescence in situ hybridization (QFISH) is an approach combining fl uorescence in situ hybridization (FISH) and digital quantifi cation of microscopic images. It has been shown to be applicable for a variety of purposes in molecular cytogenetics studies [ 1 – 4 ]. QFISH represents an important part of studying somatic chromosomal mosaicism and molecular cytogenetic detection of chromosomal variations in interphase nuclei [ 5 – 9 ]. Furthermore, QFISH using DNA or peptide nucleic acids (PNA) probes has been successfully employed for the in situ quantifi cation of telomeric DNA repeats [ 1 , 10 , 11 ]. This technique is then subsequently devel- oped for distinguishing the two homologous human chromosomes of parental origin at single cell level [ 12 , 13 ]. Strikingly, QFISH are found to be an important and indispensable tool in cancer research nowadays. In this context, these applications have been proven to enhance the effi ciency for the detection of chromosome instability and quantifi cation of gene amplifi cation or correlation of the size of noncoding repeated sequences during disease progression [ 7 , 11 ,
14 – 16 ]. Additionally, these approaches have also been used for the discrimination and quantifi cation of microorganisms in microbiol- ogy studies [ 17 ].
To enhance the precision of molecular cytogenetic analysis of human chromosomes, QFISH is generally employed to discrimi- nate “real” genomic/chromosomal change from false-positive sig- nal appearance. For example, chromosome loss in interphase nuclei can be differentiated from signal overlapping using QFISH [ 3 – 9 , 18 ]. Furthermore, QFISH allows visualization of small copy num- ber variations in situ [ 19 ]. Thus, chromosome instability analysis seems to be benefi tted from QFISH [ 3 – 9 , 15 , 16 , 18 , 19 ]. Finally, QFISH has enabled developing more sophisticated FISH-based approaches, i.e., interphase chromosome-specifi c multicolor band- ing (ICS- MCB ) [ 20 , 21 ] and 3-dimensional (3-D) profi ling of FISH signals [ 22 ], which enable analyses of somatic genome varia- tions and nuclear genome organization at chromosomal (supramo- lecular) level [ 20 – 22 ]. Here, a detailed QFISH protocol from cell suspension preparation for interphase FISH [ 23 ], basic FISH pro- cedures [ 3 , 5 – 9 ] and quantitation of FISH signals using ImageJ software [ 24 ] is presented.
2 Materials
The standard molecular biological and cytogenetic equipment, including standard solutions (ethanol, methanol, formamide, formaldehyde, etc.), are not mentioned. Only specialized items are listed below ( see Note 1 ).
1. 20× Saline sodium citrate (SSC).