|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
To study novel genes that play a role in tumorigenesis and progression, we originated a new method, comparative mRNA hybridization (CmRH), which enabled us to detect visually expression levels of known and unknown genes. In this research project, we intended to establish the method of CmRH,and to know whether we could put CmRH into practical use.
This method consists of three processes as follows ;
1.getting cDNAs from both tumor sample and control one and labeling them with either digoxigenin or biotin, respectively.
2.hybridizing the labeled cDNA probes simultaneously onto a normal metaphase and detecting by either avidin-FITC or anti-digoxigenin-Rhodamine, respectively.
3.visualizing by a fluorescence microscope equipped with a charge-coupled-device camera and calculating ratio of green and red signal intensity on each locus of the metaphase.
We got data and insight as follows ;
1.When we tried to make labeled cDNA probes directly from mRNAs with oligo (dT) primer, biotin-dUTP (or digoxigenin-dUTP) and reverse transcriptase, we got too long (2-3kb) cDNA probes to use in comparative hybridization. Although, we could get optimized length (0.5-1kb) probes when we made cDNAs at first, then labeled them by nick-translation.
2.We could not get enough signal intensity, when cDNA probes were hybridized on a normal metaphase as compared with DNA probes. Intron sequence within the metaphase might be one reason we cannot get sufficient hybridization of cDNA probes to the metaphase. We have to down stringency in post-hybridization washing procedures to get enough hybridization of cDNA probes. Although, if we down the stringency, specificity of the probes might fall down. We must solve this problem before putting CmRH into practical use.