Technical Development of Molecular Profiling Technologies

分子分析技术的技术发展

• 批准号: 7733440
• 负责人: PAUL S. MELTZER
• 金额: $ 77.33万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733440
• 关键词: Anatomy; Archives; Area; Arts; Biochemical; Biological; Biological Assay; Biological Markers; Biopsy; Blood; Capillary Electrophoresis; Cell Count; Cell Cycle; Cells; Chemicals; Chemistry; Chloroform; Clinical; Clinical Investigator; Clinical Trials; Clinical Trials Design; Collaborations; Commerce; Complementary DNA; Condition; Custom; Cytology; DNA; DNA Methylation; DNA Sequence; DNA copy number; Data; Depth; Detection; Development; Disease; Drug Delivery Systems; Ensure; Face; Fluorescence; Formalin; Freezing; Future; Gene Expression; Genes; Genome; Genomics; Goals; Government; Health Hazards; Institutes; Laboratories; Leadership; Malignant Neoplasms; Medicine; Messenger RNA; Methylation; Microfluidic Microchips; Microfluidics; Mission; Molecular Profiling; Mutation; National Cancer Institute; Nucleic Acids; Numbers; Organic solvent product; Paraffin Embedding; Pathology; Phenols; Polymerase Chain Reaction; Postdoctoral Fellow; Procedures; Process; Protocols documentation; RNA; RNA amplification; Reaction; Reproducibility; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Running; Sample Size; Sampling; Science; Scientist; Site; Slide; Specimen; Standards of Weights and Measures; Students; System; Technology; Testing; Time; Tissues; Training; Warm Ischemia; Work; base; comparative genomic hybridization; epigenomics; improved; innovation; insight; nanolitre; new technology; next generation; novel; prototype; research and development; technology development; tumor; tumor growth; wasting

The Clinical Molecular Profiling Cores (CMPC) technology development efforts are primarily directed at expanding the number of clinical samples which can be analyzed. Despite the best intentions of clinical researchers, accrual of appropriate biospecimen remains the most challenging aspect of implementing the Cores personalized medicine mission. For this reason, we have directed efforts to the problem of analyzing formalin fixed paraffin embedded (FFPE) specimens. The ability to use FFPE is extremely attractive since this specimen type fits into routine pathology laboratory practices. An example of a recent project in this area is provided by our study of DNA methylation in cancer. Using a novel microarray based platform, we have established that it is possible to profile sites of DNA methylation in FFPE specimens as accurately as in frozen specimens. This will open up large archives of tissue specimens to this type of research. As expected, DNA based assays are relatively robust, however, RNA is much more labile template. We are exploring the possibility of obtaining mRNA signatures from archival material such as FFPE. This is difficult because although the platform technology is not intrinsically limiting, the fragmented RNA found in such compromised samples are subject to many variables in sample processing prior to stabilization (warm ischemia time, processing time, processing chemistry etc.) and after stabilization to varying degrees of time and conditions of storage. Despite these challenges, we believe it is worth exploring new technologies and procedures for analyzing short RNA fragments. To help ensure reproducibility and provide for quality results, we have evaluated and are continually develop standard operating procedures for extracting nucleic acid from clinical specimens. For the above mentioned study, we successfully extracted DNA from FFPE samples for use in methylation assays; significantly, these samples have been very suitable for comparative genomic hybridization and DNA sequencing. Very recently, we have developed a protocol for extracting DNA from cytology slides and have been able to generate remarkably high quality DNA copy number and mutation profiles from this material. Organic solvents such as phenol and chloroform have been used for decades to purify nucleic acids from blood and tissues. However, the use and waste produced with these chemicals creates health hazard issues and problems of disposal. Therefore, we have investigated and validated new protocols for extraction of DNA, RNA, and microRNA from both research and clinical specimens without the use of organic solvents. These efforts are illustrative of our commitment to extend the utility of genome profiling technologies to realistically obtainable clinical samples. A common problem the CMPC faces is that many of the specimens received are biopsies containing relatively few numbers of cells as compared to anatomic specimens. Also, sometimes specimens are in high demand for multiple uses and must be divided into very small amounts. Therefore, the Core is working to implement whole genome amplification prior to bringing the sample to testing on our commonly used assays such as DNA sequencing. Data have shown that these amplified sequences are suitable for DNA sequencing, but have generated problems for epigenomic assays such as methylation determination. The work is on going to make the best use of such small sample amounts and when successful will allow the CMPC to either decrease the amount of specimen needed up front or allow us to make use of what would be normally unacceptable starting amounts of very rare and precious specimens. In keeping with our goal to improve the utility of small amounts of sample and yet maximize the amount of information we can derive from such a specimen, an important project of the CMPC is to develop multiplex expression assays. Clinical investigators can utilize these to analyze specimens for particular biomarkers associated with a specific disease they are studying. For example, we are working on a cell cycle reverse transcriptase PCR assay that incorporates some 25 different genes. The amplicons from the reaction are run on the Beckman Coulter GeXP system, a capillary electrophoresis platform that uses fluorescence for detection and quantification. We are evaluating the results from this prototype assay in comparison to other multiplex platforms such as quantitative real-time PCR and the Panomics QuantiGene assay. The CMPC is actively engaged in an inter-government department collaboration developing state of the art microfluidic assays with the National Institute of Standards and Technology (NIST), part of the U.S. Commerce Department. Specifically, in partnership with the NIST Biochemical Science Division we are developing custom fabricated microfluidic devices and procedures for the nanoliter cDNA synthesis and amplification of RNA. Our goal is to reduce sample size down to single cell equivalents for analysis of gene expression. An important new area of technology development is in the application of "next generation" sequencing technologies to cinical specimens. These new technologies offer the possibility of generating genomic profiling data on tumor specimens in a much deeper and more robust way than has been possible with microarrays. For example, it may become possible to profile large numbers of drug targets for mutations which may promote tumor growth, data which could be incorporated into future clinical trials design. The leadership of the CMPC understands that training of new students and scientists will be crucial to this new field of personalize cancer medicine and to that end have actively trained in this year alone one post-baccalaureate, one post-doctoral fellow, and two summer students.