Department of Clinical Oncology

Research Facilities of LCG

Chromosome microdissection

Chromosome microdissection has been developed into a useful and reproducible means to proceed rapidly from cytogenetic observation to molecular analysis. It is carried out in two main steps. First, a sample of DNA is obtained from a cytogenetic specimen by dissecting target chromosome region under the microscope with a fine glass needle. Second, the DNA samples are modified so that they can be propagated. Here we focus on the procedures of chromosome microdissection.

Comparative genomic hybridization (CGH)

CGH is a molecular cytogenetic technique for the comprehensive analysis of DNA copy-number gain or loss across the whole genome. As it posses the sensitivity of the in situ technique and overcomes many drawbacks of conventional cytogenetic analysis, CGH is widely used as a valuable tool for the detection of chromosomal imbalances in a wide range of tumour samples. Briefly, equal amount of tumour (test) and normal (reference) DNA is labelled with different fluorochromes. The DNA is mixed together and hybridized onto the normal metaphase. The intensity of the fluorescence bound on the metaphase is characterized. The aberrant chromosomal region is determined by the calculated fluorescence ratio of the two fluorochromes. Four experimental sections are divided: normal metaphase preparation, DNA probes labelling, in situ hybridization and data acquisition.

Fluorescence in situ hybridization (FISH)

FISH is a very widely used technique on not only cytogenetic studies, but also other biological fields. It include metaphase & interphase FISH

Proteomics

Proteomics is the study of protein profiles in a given type of organism, tissue or cell. Unlike genomics, which is a fixed feature of an organism, proteomics is rather dynamic. It changes with different developmental stages, tissue types, environmental conditions, etc. Because of the limitations in tools and technologies for proteome analysis, the development of protein profiling has fallen far behind nucleotide-based analysis. With the establishment of the first human genome sequence and annotation draft and several other genome sequencings undergoing, the existing information gap between genomics and proteomics is becoming increasingly evident. Fortunately, breakthroughs in 2D gel electrophoresis and development in other protein separation technologies, in combination with advances in bioinformatics, have enabled large scale protein profiling. Comparison between proteomic maps under healthy and diseased, treatment and non-treatment conditions may allow us to understand and monitor disease process, to identify markers of diagnostic and prognostic value, and ultimately to develop both preventive and therapeutic strategies.

SHORT PROTOCOL OF PROTEOMIC STUDIES :

Sample preparation
1st dimensional iso-electric focusing (IEF)
2nd dimensional polyacrylamide gel electrophoresis(2-D PAGE)
Imaging and analysis
Protein identification and characterization

Single stranded conformation polymorphism (SSCP)

SSCP is a fast and simple technique which widely used for mutation detection in various diseases. Basically, an interested fragment is amplified by PCR (Polymerase Chain Reaction) from either genomic DNA or cDNA first, followed by electrophoresis in non-denaturing environment. The mutant DNA is separated from the normal due to the difference in mobility in electrophoresis, which is believed to be caused by the conformational change of the single stranded mutant DNA. Usually the DNA fragment size is restricted to less than 200bp as the sensitivity of PCR-SSCP is decreased if the fragment is large.

Tissue microarray (TMA)

Tissue microarray (TMA) is a high-throughput technology which facilitates the analysis of molecular alterations in thousands of tissue specimens in a massively parallel fashion. Construction of TMAs is achieved by acquiring cylindrical core specimens from up to 1000 fixed and paraffin-embedded tissue specimens and arraying them at high density into a recipient TMA block. Hundreds (100 to 300) of consecutive sections can be cut from each TMA block and probed with detection reagents for a variety of molecular targets either at the DNA, RNA or protein level.

The principle of TMA

Cylindrical core biopsies are obtained from up to 1000 individual, formalin-fixed, paraffin-embedded tissue blocks. These are transferred to a TMA block. Multiple TMA blocks can be generated at the same time. Each TMA block can be sectioned up to 300 times. All the resulting TMA slides have the same tissues in the same coordinate positions. The individual slides can be used for a variety of molecular analyses, such as H&E staining to ascertain tissue morphology, mRNA ISH or protein immunostaining or analysis of genetic alterations using FISH.

Advantages of TMA

1. A single TMA experiment can yield information on the molecular characteristics of up to 1000 specimens at once. This is in contrast to conventional analyses, where each slide contains a section of a single tissue.
   

2. The analyses carried out on TMAs also provide information on the cellular origin of the molecular targets, thereby extending the information available from gene expression microarrays.
   

3. Construction of TMAs is usually performed from archival formalin-fixed tissue materials. The ability to use archival specimens in high-throughput molecular analyses is a significant advantage.
   

4. This sample tracking system can be linked to a database containing the demographic, clinico-pathological and survival data of the patients, allowing one to rapidly link molecular data with clinical features.
   

5. Using TMA to punch multiple small cores from different regions captures the heterogeneity of the tumors more effectively. Core sampling from different tumor blocks of the same patient, perhaps including metastatic sites, may improve the sampling efficiency of TMAs beyond what can be achieved with a single section of one tumor.