1. PCR-agarose gel electrophoresis results: The experimentally designed primers amplified three Myc genes. After PCR amplification, the DNA of laryngeal cancer tissue and normal tissue cells can be seen on the agarose gel. 2 electrophoretic bands less than 300 bp. The first band is a synthetic band of two DNA fragments, N-myc (230 bp) and L-myc (227 bp). Since the difference between the two is only 3 bp, agarose gel electrophoresis cannot separate them and show one band; The second electrophoresis band is the C-myc gene (244 bp). Scan the negative film of the electrophoresis results with a laser scanner to obtain the peak area values ??of the two bands. The C-myc band of normal tissue cells is weaker than the synthetic band of N-myc and L-myc, and the ratio is less than 1. The average C×(N+L) value of normal tissue cells is 0.6.
C-myc amplification multiple = C×(N+L)×0.6 of laryngeal cancer. Considering the comprehensive factors in the experiment, it is determined that there is C-myc amplification of more than 1.5 times. The results showed that 42% of laryngeal cancers had C-myc amplification, while normal tissue cells did not have C-myc amplification.
2. PCR-neutral polyacrylamide gel electrophoresis results: Due to the DNA separation characteristics of neutral polyacrylamide gel electrophoresis, the Myc gene electrophoresis band after PCR amplification showed 3 bands. The first band is L-myc (227 bp), the second band is N-myc (230 bp), and the third band is C-myc (244 bp). The gel slice with the specific Myc gene electrophoresis band amplified was directly scanned on the laser scanner to obtain the peak area value of the three bands. Then divide the respective peak area value by the respective base number as the single copy number of the gene. Set the smallest single copy number among L-myc, N-myc and C-myc as 1, and compare the other Relative multiples of 2 genes. The mean value of L-myc:N-myc:C-myc in normal tissue cells is 1.0:2.2:3.8. Results Among the 32 cases of laryngeal cancer, 47% had L-myc and C-myc amplification, and 41% had N-myc amplification. The amplification rate of C-myc was basically consistent with the results detected by PCR-agarose gel electrophoresis (χ=0.254, P>0.614). 1. Specimens: 32 cases of laryngeal cancer tissues were obtained from inpatients in the Otolaryngology Department of our hospital, 7 cases of normal laryngeal tissues were obtained from non-cancer patients hospitalized in our department during the same period, and 3 cases of normal white blood cells were obtained from human whole blood from the blood bank of our hospital. All specimens were pathologically confirmed. According to the different degrees of pathological differentiation of laryngeal cancer, it is divided into highly, moderately and poorly differentiated cancers.
2. PCR primers: 5′-CCCAGCGAGACATCTGGAAGAA-3′, 5′-GAGAAGCCGCTCCACATGCAGTC-3′. Three genes of the Myc gene family were amplified, namely C-myc (244 bp), N-myc (230 bp), and L-myc (227 bp), which were synthesized by the Institute of Genetics of Fudan University in Shanghai.
3. Reagents: TaqDNA polymerase and dNTP are products of Promega Company; Proteinase K is a product of Merke Company; Silver Nitrate is a product of Shenyang Reagent Factory No. 2; Marker PUC 18 plasmid DNA Hea III fragment is a product of Sigma Company . 1. Template DNA extraction: refer to the method in reference [3].
2. PCR amplification: The total reaction volume of PCR amplification is 50 μl, using 50 ng of template DNA, operating on a DNA amplification instrument (Perkin Elmer Cetus), with a cycle of 94°C for 1 minute and 55°C 1 minute, 72°C for 2 minutes, and after 30 cycles, add 72°C for 7 minutes.
3. Agarose gel electrophoresis step: Take 5 μl of PCR product and add 1 μl of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanide FF, 40% sucrose aqueous solution) and set aside. Prepare a 2% agarose gel and pour it into a horizontal electrophoresis tank. After the gel solidifies, add the sample, use 1×TAE buffer as the electrode buffer, and stain with ethidium bromide. The voltage should be lower than 5V/cm for about 2 hours. After electrophoresis, the gel was directly observed on a UV transilluminator. After taking pictures, the negatives are scanned on a laser scanner.
4. Non-denaturing polyacrylamide gel electrophoresis step: Take 5 μg of PCR product and add 1 μl of loading buffer and set aside. Prepare an 8% nondenaturing polyacrylamide gel and pour it into a 20 cm × 20 cm × 0.1 cm vertical plate electrophoresis tank. After the gel polymerizes, add samples, use 1×TBE buffer as the electrode buffer, and store at room temperature. Electrophoresis was carried out at 1 watt for about 14 hours, and the electrophoresis was stopped when the indicator bromophenol blue moved to the edge. The gel after electrophoresis was transferred to a square plate for silver nitrate staining.
The staining process is as follows: wash the gel 3 times with double-distilled water (dH2O), then soak it in the staining solution (1g silver nitrate, add dH2O to 500 ml) for 30 minutes, remove the staining solution, rinse the gel 3 times with dH2O, and then soak it in Put it in the chromogenic solution (NaOH 15g, 38% formaldehyde 3.8 ml, add dH2O to 500ml) for about 10 minutes. When a brown-black DNA band appears, wash the gel once with dH2O and then immerse it in the stop shadow solution (glacial acetic acid 25 ml, add water to 500 ml) for about 30 minutes, then move the gel to the fixative (25 ml of ethanol, 2.5 ml of glacial acetic acid, add dH2O to 500 ml) for fixation. The fixed gel is observed on the film viewing lamp, photos are taken, and the gel is scanned directly on the laser scanner.
The three genes have two regions that are highly homologous in the second exon. The three genes express independent proteins, and their physiological effects are basically the same. Studies have shown that the Myc gene is amplified or abnormally expressed in many tumor cells, but in different tumor tissues and cancer cell lines, members of the Myc gene family have some differences in amplification or expression. Therefore, it is of great significance to further study the relationship between the three members of the Myc gene and human tumors. The PCR-nondenaturing polyacrylamide gel electrophoresis-laser scanning technology established in this study can simultaneously detect the amplification of three Myc genes in tumor tissues. During the development of tumors, it is necessary to understand the respective mechanisms of action of the three genes. Research provides simple experimental means.