Presentation on theme: "Laser microdissection MCB 1 st April, 2013. Purpose To allow us to utilise material that cannot be obtained by other methods To select only the cells."— Presentation transcript:
Laser microdissection MCB 1 st April, 2013
Purpose To allow us to utilise material that cannot be obtained by other methods To select only the cells or tissue that we want
Overview of LMD Excise tissue (cells) from tissue Dissection under microscope Exclude contaminating cells/materials Many potential tissue sources Accurate discrimination (staining, hi-mag)
Honaas et al. Functional genomics of a generalist parasitic plant: Laser microdissection of host-parasite interface reveals host-specific patterns of parasite gene expression. BMC Plant Biology 2013, 13:9 Example of fine selection
Downstream applications QPCR, RNA-seq, microarray for gene expression analysis Protein isolation DNA sequencing for tissue specific sequences More detail in the next laboratory presentation
Technical overview Attach tissue to membrane (PEN, etc.) Dissect out desired tissue with laser LMD is not the same as LCM. Laser Capture Microdissection Emmert-Buck et al., 1996
Laser power source Microscope X-Y and objective controls Microscope software The microscope
Sectioning the tissue Figure 1. Laser microdissection enables profiling of gene expression at different stages of leaf development. Morphogenetic primordia (A, 750μm) and from the shoot apical meristem (remaining in C). In addition to classification of primordia by length, the area of the tissue excised is recorded. Tissues for each stage were pooled for RNA extraction using an Arcturus Paradise kit. Randomly primed reverse transcription has yielded cDNA for quantitative PCR comparison of development-associated candidate gene expression. Satisfactory yields were obtained from approximately μm tissue sections. ABC
Harvest tissue Fix/preserve tissue Mount tissue on membrane Deparaffinise Stain material for identification ✓ ✓ ✓ ✓ Dissect tissue Downstream processing Plant shoot tips – mature leaf and stem, young leaf and SAM 3:1 ethanol:acetic acid Leica PEN membrane slides Ethanol/ xylene; 100% ethanol; 95% ethanol; 70% ethanol E.g. eosin, haematoxylin Genomics Facility LMD 6000 RNA, DNA, protein, carbohydrates, etc. (Heat block)
Protocol-specific decisions Tissue type, quality and quantity Fixation method - Paraformaldehyde; acetone; ethanol/ethanol:acetic acid; methacarn (methanol, chloroform and acetic acid) - Affects morphology and downstream processes Cryogenics or paraffin embedding - Preservation of molecules or morphology Sectioning - Section thickness; arrangement, pooling Additional steps - RiboAmp?
References Leica LMD home - microdissection/http://www.leica-microsystems.com/products/light-microscopes/life-science-research/laser- microdissection/ Leica LMD preparation protocol - Fixation methods - Kim, J.-O., Kim, H.-N., Hwang, M.-H., Shin, H.-I., Kim, S.-Y., Park, R.-W., Park, E.-Y., Kim, I.-S., Van Wijnen, A.J., Stein, J.L., Lian, J.B., Stein, G.S., Choi, J.-Y. Differential Gene Expression Analysis Using Paraffin-Embedded Tissues after Laser Microdissection (2003) Journal of Cellular Biochemistry, 90 (5), pp Plant tissue fixation - Kerk, N.M., Ceserani, T., Lorraine Tausta, S., Sussex, I.M., Nelson, T.M. Laser capture microdissection of cells from plant tissues (2003) Plant Physiology, 132 (1), pp Example - Honaas, L.A., Wafula, E.K., Yang, Z., Der, J.P., Wickett, N.J., Altman, N.S., Taylor, C.G., Yoder, J.I., Timko, M.P., Westwood, J.H., dePamphilis, C.W. Functional genomics of a generalist parasitic plant: Laser microdissection of host- parasite interface reveals host-specific patterns of parasite gene expression (2013) BMC Plant Biology, p. 9. Article in Press. Arcturus Paradise kit manual - LCM method - Laser Capture Microdissection for Analysis of Gene Expression in Formalin-Fixed Paraffin-Embedded Tissue. Ru Jiang, Rona S. Scott, and Lindsey M. Hutt-Fletcher Overview with notes for hepatology, protein, RNA and DNA - Laser Microdissection Microscopy and its Applications in Molecular Biology. Ahlam Mustafa, Ragai R Mitry and Alberto Quaglia, 2010 Acknowledgements Great thanks to Vijay Nadella at the Genomics Facility (5 th floor, Porter Hall). Useful discussions were had with Dr Faik and with Loren Honaas from Penn. State.