Edited by Xian-Lin Wang
Proofread by Jing-Yun Ma
Messenger RNA differential display technique, developed by Dr. Liang in 1992, is a powerful new tool for identifying and cloning differentially expressed genes in a certain type of cell line, tissue or a special developing stage. Using this method, large amounts of molecular biological information can be obtained easily and quickly. In our study, this technique was used to compare genes expressed differentially between normal colon mucosa and colon carcinomas, in order to understand the molecular biological basis of colon cancer.
Fifteen samples of colon carcinoma were obtained from radical colectomy, samples of normal colon mucosa were obtained 8 cm-10 cm apart from colon carc inoma.
Guanidine thiocyanate and β-mercaptoethanol were purchased fro m BRL (America); T12MN primer and AP primer were presented by the National Laboratory for Oncogene and Related Genes, Shanghai Cancer Institute.
Reverse transcription DNA-free RNA was reversely transcribed using the oligo-dT primer T12MN in the presence of [γ-32P]dATP.
PCR amplification Using cDNA products as temples, PCR reaction s were performed in the presence of [γ-35S] dATP, the primer combination was T12MN and AP. Following denaturation for 30 seconds at 94 °C, the PCR steps consisted of 30 seconds at 94 °C, 2 min at 40 °C, 30 seconds at 72 °C for 40 cycles, followed by 5 min at 72 °C. Amplified PCR products from normal colon mucosa and colon carcinomas were separated side by side on a 7.5 M urea/6% polyacrylamide gel.
Recovery of differentially expressed bands After autoradiograp hy, the cDNA bands representing differently expressed mRNAs were excised from the gel. For each band, extracted cDNA was reamplified for 30 cycles with the same primers and the same PCR conditions used in the initial PCR, except that no radioactive dNTP was included. After PCR, the product was run on 1.5% low melt agarose gel and stained with ethidium bromide.
Northern blot analysis PCR bands of the expected size were cut from the gel, purified and used as probes. DNA probes were radio-labeled by the random prime labeling method, hybridized with RNA from pre samples and other RNA samples respectively.
Total RNAs from normal colon mucosa and colon carcinomas were isolated using single-step method of guanidine thiocyanate, then reversely transcribed into first string of cDNA, T12MA, T12MC, T12MT and T12MG were used as oligo-dT primer individually. The result of alkaline denatured electrophoresis indicated that cDNA molecules were in the range of 0.5kb-5kb. cDNAs were amplified by PCR reaction using the primer combination. T12 MN and AP2, AP4, PCR products were separated by 6% polyacrylamide gel electrophoresis. Fourteen bands were obtained which were differentially displayed between normal colon mucosa and colon carcinoma. Eight bands (T1-T8) were highly expressed in carcinomas, and the other 6 bands were expressed only in normal tissues. T1 band was verified to be highly expressed in tumor, but had no expression in normal tissues by Northern blot. This cDNA band would be used for cloning and sequencing.
The technique of mRNA differential display, by means of combinating T12MN and arbitrary primer AP, can detect all expressed genes in mammalian cells and recover their molecular biological information. These cDNA fragments can be used as probes to isolate target genes from genomic DNA or cDNA library for intensive molecular biological identification. The technique has several advantages over other methods, such as simplicity, sensitivity, reproducibility, versatility and speed, so that it has been used in researches of many diseases, especially in molecular biological study of malignant tumors. We screened 14 cDNA fragments by means of mRNA DD, one of these bands (T1) was highly expressed in the colon carcinoma which was used for differential display. Using T1 band as probe, we found that it was also highly expressed in many other colon carcinomas ( 12/15 ). In the further study, this DNA fragment can be used for cloning and sequencing. Checking the database of Genebank, if the sequence of this cDNA band has no homology to the sequences of other nucleic acids, it can be considered as partial cDNA fragment of a new colon carcinoma-related gene. By screening the genomic DNA or a certain cDNA library, the full-length cDNA can be cloned, which may be helpful in the study of the molecular biology of colon carcinoma.
Edited by Xian-Lin Wang
Proofread by Jing-Yun Ma
|1.||Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992;257:967-971. [PubMed]|
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