Multicolor fluorescence in situ hybridization assay
Multicolor fluorescence in situ hybridization assay
M-FISH has been successfully used to identify marker or derived chromosomes in congenital diseases. Recently, this method has been increasingly used to identify multiple chromosomal abnormalities in complex karyotypes; M-FISH is useful in detecting critical chromosomal rearrangements in the diagnosis of hematologic disorders.
Operation method
Multicolor fluorescence in situ hybridization assay
Materials and Instruments
DAPI Restaining Solution Ethanol SSC Hydrochloric Acid HCl NP-40 Trypsin NaCl Sodium Citrate Spectra Vysion Probe M-FISH Probe Move I. Chromosome specimen preparation 1. Specimen preparation: samples from different specimen sources (blood, amniotic fluid, fibroblast cultures or bone marrow) are prepared using their corresponding standard procedures. 2. A phase contrast microscope is used to locate the appropriate intermediate chromosome split phase and the hybridization region is marked out. 3. Stand the specimen in a staining vat with 40 mL of trypsin solution at 37°C for 2 min. 4. The slices were artificially aged in a HYBrite (Vysis) unit at 90°C for 2 min. 5. Rinse the slices in a HYBrite(Vysis) device with 2XSSC for 2 min. 6. The specimens were dehydrated for 1 minute at room temperature in a staining vat containing 40 mL of 70%, 85% and 100% ethanol, respectively, and air-dried. 1. Add M-FISH probe mix (Vysis) to the labeled hybridization region. 2. Cover the hybridization area of each specimen with a cover slip and seal the piece with rubber cement around the cover slip. 3. Fix the specimens in a humidified HYBrite (Vysis) unit set to denaturation temperature of 80°C for 3 min and hybridization temperature of 37°C for a minimum of 16 h. The specimens are then incubated in the HYBrite (Vysis) unit at a temperature of 80°C for 3 min. 4. The specimens were washed in a staining vat containing 40 mL of 0.4XSSC at 70°C for 1?2 min, and then washed for 2 s with running deionized water. 5. Transfer the specimen to a staining vat containing 40 mL of 2XSSC/0.1% NP-40 solution and apply IOs06 at room temperature. Add 10% DAPI compound to each hybridized area and cover the slides, taking care not to seal the slides. Caveat 1. Regardless of whether the specimen is freshly prepared or the specimen is M-FISHed after banding, the M-FISH probe (Vysis, DownerGrove, IL) is added to the hybridization region, the coverslip is covered and sealed with rubber cement, and the slide is placed into wet Hybrite with the following settings: unchaining temperature of 80°C for 3 min, and hybridization temperature of 37°C for at least 16 h. The results are usually better in freshly prepared specimens because G bands are usually artificially aged. M-FISH hybridization results are usually better for freshly prepared specimens because the G-bands are usually artificially aged. 2. An important point to consider when performing M-FISH analysis is the contrast between hybridization and non-specific binding at the target site. If the contrast is poor, the chromosome sorting colors will also be unremarkable. Contrast is especially important for far-infrared fluorescence. Longer trypsin processing times will help improve contrast, while too long a processing time will result in loss of chromosomal DNA and signal, which will result in darker chromosomes. Insufficient humidity in the hybridization cassette will dehydrate and concentrate the hybridization reagents, which will result in random hybridization (loose binding) of the probe and chromosomal DNA, which will result in a loss of contrast. 3. Another important factor in obtaining favorable results is chromosome morphology. Poor chromosome morphology will make it difficult to interpret DAPI bands when converted to grayscale, and can also cause chromosome color to flow from one chromosome to another. The most common cause of poor chromosome morphology is inadequate aging, artificial aging or treating the slice with hot 2XSSC for 2min helps to improve chromosome morphology. Usually rinsing the specimen with deionized water at room temperature followed by DAPI restaining also improves banding but decreases hybridization intensity. Increased aging or longer 2XSSC pretreatment times may also reduce hybridization intensity. Poor chromosome morphology can also be caused by excessive denaturation. Good chromosome morphology but poor hybridization is usually indicative of over-aging, prolonged 2XSSC treatment, or insufficient denaturation of the specimen. Results can also be adversely affected by dirty specimens that are prepared and leave a lot of background debris on the slide. Bright fluorescent debris will shorten the exposure time and affect chromosome sorting, which is especially noticeable with Texas Red. This problem can be solved by lengthening the exposure time of the specimen to the Texas Red plane in manual mode. 4. Sometimes it is difficult to obtain optimal results even after the best efforts, reagents or procedures are correct. It is then necessary to look at each light plane separately and compare it with the table provided by the manufacturer. For example, chromosome 22 (light green, red, and green) is sometimes misclassified as chromosome 3 (light green), and it is possible to determine whether the observed chromosome is chromosome 22 by looking at the red and green planes of light separately to determine whether these two spectra are present. 5. In reciprocal translocations between chromosomes and chromosome overlaps, chromosome differentiation at the junction of breaks and fusions is often suggested by the presence of a third chromosome or by the fact that the region turns gray due to the overlap of fluorescein with each other (Fig. 50.) The above scenario is important when the translocated fragments are very small, and all possible chromosomes are taken into account based on the observed color classification. For example, a derived chromosome 7 with a fragment translocated from chromosome 12 might be classified as chromatin 19 (Fig. 5D) because chromosome 7 is labeled far-infrared (farred), and 12 is gold and green, so the three color combinations (far-infrared, gold, and green) happen to be characteristic of chromosome 19. Therefore to solve this problem, we have to analyze the samples with the chromosome 12 wcp probe, and a positive result will conclude that the source of the translocated fragment is chromosome 12, and a negative result will conclude that the source of the fragment is chromosome 19. Fluorescence combinations also appear as gray areas when they are outside the classification system, e.g., an overlap of chromosomes 20 (far-infrared, light green, and red) and 21 (golden yellow and light green) results in a 4-color combination (far-infrared, light green, red, and golden yellow), which would appear as a gray area, since no chromosome has more than 3 color combinations. 6. Small translocations, especially interpolators, are particularly susceptible to misinterpretation when the fluorescence spectrum of the derived chromosome contains all the fluorescein features of the translocated chromosome. As an example, when a small fragment of chromosome 1 (golden yellow) is translocated to chromosome 5 (golden yellow and far infrared), the whole chromosome still looks like chromosome 5. The use of a chromosome 1 coating probe alone can solve this problem. 7. Constitutive heterochromatin, including segments rich in a-satellites and short proximal mitotic arms rich in repetitive DNA sequences, can be closed off with unlabeled Cot1-DNA or probed without their complementary sequences. These regions also turn gray or are randomly pseudocolored and sorted (Fig. 5C), a phenomenon that must be kept in mind when analyzing labeled or derived chromosomes.Some other limitations of M-FISH include the inability to detect most intrachromosomal anomalies (e.g., inversions) and interchromosomal anomalies no larger than 3 Mb. For more product details, please visit Aladdin Scientific website.