1 Laser Microdissection Analysis Platform Administrative Coordination: M. Nakache (CR1) Scientific Coordination: T. Jay (DR2) & L. Villanueva (DR2) Experimental.

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Presentation transcript:

1 Laser Microdissection Analysis Platform Administrative Coordination: M. Nakache (CR1) Scientific Coordination: T. Jay (DR2) & L. Villanueva (DR2) Experimental Coordination: E. Davenas (IR1)

2 Laser Microdissection : a tool within a process Extraction (2) Histologic Observations Histochemical-immunity In Situ Hybridation TUNEL (4) Laser Microdissection Genome Transcriptome PCR RT-PCR Proteome Western blot 2D-gel, 2D-DIGE Mass Spectrometry Tissue Prelevement (1) (3) localisation

3 Goals Cell or tissu isolation under morphologic control in order to perform molecular biology studies from either frozen or paraffin-embedded tissue slices or cell cultures

4 From tissue to molecule (1) 1. Sample preparation and cutting - fixation - paraffin embedding - freezing 2. Visualisation of areas of interest - staining - fluorescence 3. Selective Microdissection - no contact, no contamination - any size (µm 2 -> mm 2 ), shape, thickness (< 150 µm)

5 LMPC PALM® System Laser Microdissection & Pressure Catapulting The laser beam cuts the area of interest then, by adjusting its energy and its focusing, it catapults the selected region into the cap of a tube placed just over. This cap is filled with a lysis buffer adequate for subsequent analysis: - DNA - RNA - Protein

6 From tissue to molecule (2) 4. Extraction and preparation of specific molecules: DNA, RNA, proteins 5. Analysis of pertinent, reproducible and specific results

7  DNA Extraction  Tumor Analysis  DNA Fingerprinting DNA RNA  RNA Extraction  Quantitative RT-PCR  Microarray analysis Protein  HPLC  SDS-PAGE  Mass Spectroscopy analysis (MALDI, SELDI) Molecular analysis: DNA, RNA, Proteins Molecular analysis: DNA, RNA, Proteins

8 Materials available PALM® Microbeam Laser Microdissector (Zeiss) Cryostat HM550 (MM France) Microtome HM450 (MM France) UV Crosslinker BLX-254 (BIO-LINK) Hood Chemfree 90S (Oxygen)

9 Laser Microdissection 2,5X 10X 2,5X 10X 2,5X 10X Globus Pallidus Globus Pallidus Subthalamic Nucleus Subthalamic Nucleus Black Matter Black Matter