2 PurposeTo allow us to utilise material that cannot be obtained by other methods To select only the cells or tissue that we want
3 Overview of LMD Excise tissue (cells) from tissue Dissection under microscopeExclude contaminating cells/materialsMany potential tissue sourcesAccurate discrimination (staining, hi-mag)
4 Example of fine selection 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
5 Downstream applications QPCR, RNA-seq, microarray for gene expression analysisProtein isolationDNA sequencing for tissue specific sequencesMore detail in the next laboratory presentation
6 Laser Capture Microdissection Technical overviewAttach tissue to membrane (PEN, etc.)Dissect out desired tissue with laserLMD is not the same as LCM.Less heating of tissue with LMD versus LCM.Laser Capture MicrodissectionEmmert-Buck et al., 1996
7 X-Y and objective controls The microscopeMicroscope softwareLaser power sourceX-Y and objective controlsMicroscope
8 Sectioning the tissue A B C Figure 1. Laser microdissection enables profiling of gene expression at different stages of leaf development. Morphogenetic primordia (A, <750μm) were excised separately from maturing primordia (B, >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
10 Plant shoot tips – mature leaf and stem, young leaf and SAM Harvest tissue✓Plant shoot tips – mature leaf and stem, young leaf and SAMFix/preserve tissue✓3:1 ethanol:acetic acidMount tissue on membraneLeica PEN membrane slides(Heat block)DeparaffiniseEthanol/ xylene; 100% ethanol; 95% ethanol; 70% ethanolStain material for identificationE.g. eosin, haematoxylinDissect tissueGenomics Facility LMD 6000Downstream processingRNA, DNA, protein, carbohydrates, etc.
11 Protocol-specific decisions Tissue type, quality and quantityFixation method - Paraformaldehyde; acetone; ethanol/ethanol:acetic acid; methacarn (methanol, chloroform and acetic acid) - Affects morphology and downstream processesCryogenics or paraffin embedding - Preservation of molecules or morphologySectioning - Section thickness; arrangement, poolingAdditional steps - RiboAmp?
12 References Acknowledgements Leica LMD home -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), ppPlant 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), ppExample - 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-FletcherOverview 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, 2010AcknowledgementsGreat thanks to Vijay Nadella at the Genomics Facility (5th floor, Porter Hall).Useful discussions were had with Dr Faik and with Loren Honaas from Penn. State.