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Ref: Laboratory Investigation. 2001 Apr;81(4):475-81.Get article in PDF format here
CM-FISH Fig 1 & Table 1-legend
a. Combinatorial and ratio labeling example: simultaneous use of three probes, pZ16a (FITC), pC1.8 (Cy3) and pG-A16 (Cy5) allows detection of four centromeres (chromosomes 1, 5, 16 and 19). Individual channel images (grayscale) for this hybridization are shown in Fig. 2 a-c. b. Partial G-banded metaphase of a case with a small, unidentified marker chromosome (47, XX, + mar). c. CM-FISH analysis of a metaphase from the same case. The gray-scale images of the six channels (AMCA, DEAC, FITC, Cy3, Cy5, Cy5.5) were combined and pseudocolored using the PowerGene M-FISH software (PSI, Inc). Because various probes label chromosomes with different intensities, the imaging software merging and pseudocoloring the channels may display signals from unrelated chromosomes with similar color. Therefore, unlike regular M-FISH, the marker is not identified by its color. Rather, after its position in the metaphase is identified (d), the marker is examined in all six channels and identified by its labeling pattern (Table 1). For one chromosome in every pair, table 1 displays the hybridization signal in each of the six fluorescence channels separately. The 47th signal (15, arrow) in the metaphase (c) matches perfectly the pattern of hybridization corresponding to the two chromosomes 15, indicating that the marker is of chromosome 15 origin. d. When using the fluorophore AMCA, DAPI counterstaining is not possible. However, overexposure of the same metaphase in the DEAC channel (2-4 seconds), clearly shows position of the small marker chromosome without requiring DAPI. e. Interphase CM-FISH analysis using mix C1 on a bone marrow sample with trisomy 8. f. Black and white image of the same nucleus, with numbers indicating the position of every centromere. Individual channel images for this nucleus are depicted in Fig. 2 (d-i).
Table 1. CM-FISH combination algorithm. The columns in the table depict the six fluors/haptenes used to label the probes. Numbers in the gray columns indicate the chromosome numeral/name. For each numeral, the corresponding row shows the number of microliter of the nick translation labeling reaction (10 ng/m l DNA) used for one hybridization. These numbers show that some chromosomes require much less probe than others to be detected. The occasional small black dots indicate that, for the respective channel, the centromere probe of a different chromosome will cross-hybridize there, and is part of the detection algorithm. For example, the black dot in the Cy3 channel for chromosome 1, indicates that this centromere will acquire a Cy3 hybridization, coming from a different centromeric probe, in this case pC1.8. In the gray columns, under each number the table displays the colored CM-FISH image of the respective chromosome, as it appears in Figure 1c. The row corresponding to every colored signal, depicts the hybridization pattern in each of the six fluorescence channels. The light-yellow cells indicate the channel(s) in which the respective chromosome is expected to show hybridization signal. The signals can but DO NOT have to be of similar strength for the analysis to work, as for those chromosomes ratio labeling is not important (for example, chromosome 12). An empty yellow cell indicates that the respective signal is sometimes absent, but does not affect chromosome identification. The light-red cells, indicate that ratio labeling is important for those chromosomes, and that the signal corresponding to the respective channel is the strongest. For example, chromosomes 4 and 9 both show Cy5 and DIG hybridization signals, but Cy5 is stronger on chromosome 4, whereas DIG/DEAC is stronger on 9. As the metaphase shown in (c) does not have a Y chromosome, the Y hybridization pattern was added to the table from a male metaphase. Finally, because of the related alphoid families, many chromosomes show background hybridization in "non-specific" channels. These signals are always weaker than the specific signals, are inconsistent (sometimes not present, depending on the slide quality and hybridization conditions) and do not interfere with chromosome identification.
Additional observations: Chromosome 1 acquires Cy3 signal (black dot) from pC1.8. Chromosome 5 acquires FITC from pZ16A and BIO/AMCA from pG-A16. Chromosome 16 acquires FITC signal from pZ16A. Chromosome 19 acquires Cy3 from pC1.8 and occasionally FITC from pZ16A. Chromosomes 4,9 and 2,18,20 are detected using ratio labeling. This was necessary as the centromeric probes of 4 and 9 and of 2, 18 and 20 respectively, cross-hybridize to one another to some extent. The cross-hybridization of L1.84 to chromosomes 2 and 20 is not very apparent (no DNP/Cy5.5 signals). However, probes from 2 and 20 (pBS4D and pZ20) hybridize to chromosome 18 centromere, thus the expected Cy5 and BIO/DEAC signals (black dots). Because of this, centromere 18 will have a different signature than 2 or 20 simply by adding the DNP-labeled L1.84. Thus, whereas centromeres 2 and 20 are identified by true ratio labeling, centromere 18 has a characteristic signature. Chromosomes 13 and 21 are detected with the Cy5-labeled probe L1.26 and both occasionally present a faint Cy3 signal, probably from the cross-hybridization of probe p3-9. Chromosomes 14 and 22 are both detected by the BIO-labeled µ -XT(680). Their separate identification is achieved using the DIG/DEAC labeled p22/1:2.1;2.8;0.73, which does not cross-hybridize visibly on 14.