Fluorescence in situ hybridization (FISH) is an emerging molecular cytogenetic technology, a non-radioactive in situ hybridization technique developed on the basis of the original radioactive in situ hybridization technology, which is characterized by rapidity, safety, high hybridization specificity, and the possibility of multiple staining, and therefore has received widespread attention in the field of molecular cytogenetics. Therefore, it has received widespread attention in the field of molecular cytogenetics.
Principle
In accordance with the principle of base complementary pairing, a semi-antigen-labeled DNA or RNA probe is paired with a denatured single-stranded nucleic acid sequence, which is detected by an antibody with a fluorescent group that recognizes the semi-antigen, or by directly labeling the probe with a fluorescent group that binds to the target sequence, and then finally, the distribution of the target sequence in the nucleus, chromosome, or section of the tissue is directly observed by a fluorescence microscope.
Appliance
Currently this technology has been widely used in many fields such as animal and plant genome structure studies, chromosome fine structure variation analysis, viral infection analysis, human prenatal diagnosis, tumor genetics and genome evolution studies.
Operation method
RNA-FISH
Principle
In accordance with the principle of base complementary pairing, a semi-antigen-labeled DNA or RNA probe is paired with a denatured single-stranded nucleic acid sequence, which is detected by an antibody with a fluorescent group that recognizes the semi-antigen, or by directly labeling the probe with a fluorescent group that binds to the target sequence, and then finally, the distribution of the target sequence in the nucleus, chromosome, or section of the tissue is directly observed by a fluorescence microscope.
Materials and Instruments
Reagents: Move 1、Probe design and synthesis The probes were designed according to the experimental requirements, and the designed probes were compared on NCBI to determine the specificity of the designed probes.
Probe, sample, PBS, formaldehyde, formamide
Dextran sulfate, anhydrous ethanol, 2×SSC, DAPI
Instruments:
Constant temperature water bath, incubator, slides
Confocal microscope, coverslips, sealing film
200 μL pipette, etc.
Fixation solution: 1×PBS + 3.7% formaldehyde
Wash buffer: 2×SSC + 10% formamide
Hybridization solution: 2×SSC + 100 ng/mL dextran sulfate + 10% formamide
3、RNA FISH(1) Place a clean coverslip in the center of the bottom of a 12-well cell culture dish, and then spread cells in the well for culture.
(2) When the density of the cells reaches about 70%, remove the medium, wash the cells twice with 1 mL of 1×PBS to remove the dead cells, then add 1 mL of fixative and fix for 10 min at room temperature.
(3) Remove the fixative, wash the cells twice with 1 mL of 1×PBS for 5 min each time, add 1 mL of 70% ethanol solution to cover the cells, and put them in the refrigerator at 4 ℃ for overnight permeabilization.
(4) Remove the 70% ethanol solution, add 1 mL of washing buffer, place the 12-well plate on a shaker, and wash the cells twice with shaking at room temperature and 60 rpm for 5 min each time.
(5) Take an empty 20 μL cartridge and put a clean sealing film on the headstock. Spot 50 μL of hybridization solution with probe (final concentration of 125 nM) on the sealing film, and then invert the cell side of the coverslip onto the hybridization solution, so that the cell side is in direct contact with the hybridization solution.
At the same time, 20 mL of 2×SSC solution was poured into the cassette to maintain the temperature in the cassette and to prevent volatilization of the probe solution. Seal the cassette with a sealing film and incubate it overnight at 37 ℃ in an incubator protected from light.
(6) After incubation, remove the coverslip carefully with forceps and transfer it to a 12-well plate with 1 mL of washing buffer, keeping the side with cells facing up, and incubate for 30 min under light.
(7) Remove the washing buffer, add 1 mL of washing buffer containing DAPI (final concentration of 5 ng/mL) to stain the nuclei, and incubate for 30 min, remove the washing buffer, add 1 mL of 2×SSC solution, and incubate for 10 min, protected from light.
(8) Take a clean slide and apply a small amount of sealing solution, then gently pick up the coverslip with tweezers, carefully remove the residual liquid, and invert the side of the coverslip with cells onto the sealing solution.
(9) After sealing, the slides were dried naturally for 1 h at room temperature, and finally imaged and analyzed under a confocal microscope.
Caveat
1. Sample preparation: Sample preparation should be adequate to ensure sufficient number of cells (1000~2000) and cytoplasmic integrity. For fixed cells, it is best to use fresh, high-quality cells that have been properly treated and fixed.
2. Hybridization probes: Choose appropriate hybridization probes, for example, the probe can be designed according to the sequence to be tested. At the same time, we should also pay attention to the labeling method, concentration and hybridization time of the probe and other parameters.
3. Hybridization conditions: Hybridization conditions need to be carefully controlled, including temperature, salinity, pH value, etc. Usually, the hybridization temperature is set at a temperature of 0.5°C. Normally, the hybridization temperature is 37 ℃, salinity is 2×SSC, and pH value is 7.2-7.
5.
4. Wash step: The wash step is very important to effectively remove the non-specifically bound probes and reduce the background signal. Wash buffers usually include high salt buffer and low salt buffer.
5. Color development and fluorescence microscopy: Color development and confocal microscopy need to be performed carefully to avoid misjudgment and bias. In the color development and confocal microscope observation, it is also necessary to pay attention to the signal intensity and distribution of the probe, etc..
6. Experimental safety: In the experimental process need to pay attention to safety issues, such as wearing gloves, goggles and other personal protective equipment to avoid contact with harmful substances. At the same time, you also need to pay attention to laboratory ventilation and waste disposal and other issues. Common Problems 1. Weak signal of the hybridization probe: This may be due to the low labeling efficiency of the probe or the low concentration of the probe. You can try to increase the probe concentration or change the labeling method to improve the signal strength of the probe. 2. Interference of hybridization background signal: It may be because the hybridization conditions are not ideal or the hybridization time is too long. You can try to optimize the hybridization conditions, shorten the hybridization time, or increase the washing step to reduce the background signal. 3. Probe binding non-specific: It may be because there are regions in the probe sequence that are similar to the non-target sequence, or the concentration of the probe is too high. Try to select a more specific probe or reduce the probe concentration to avoid non-specific binding. 4. Deformation of cell morphology: The cell morphology may be deformed due to improper fixation or treatment methods. Try to change the fixation or treatment method, or use more suitable samples for the experiment. 5. Uneven dispersion of probe signal: This may be due to uneven labeling of the probe or less than ideal hybridization conditions. You can try to optimize the labeling and hybridization conditions, or use more specific probes to improve signal uniformity. 6. Inaccurate data analysis: This may be due to inaccurate data analysis methods or large errors. You can try to use more accurate data analysis methods or increase the number of samples to improve the accuracy of data analysis. For more product details, please visit Aladdin Scientific website.