1. Laser microdissection
MCB
1st April, 2013

2. Purpose
To 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 microscope
Exclude contaminating cells/materials
Many potential tissue sources
Accurate 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 analysis
Protein isolation
DNA sequencing for tissue specific sequences
More detail in the next laboratory presentation

6. Laser Capture Microdissection
Technical overview
Attach tissue to membrane (PEN, etc.)
Dissect out desired tissue with laser
LMD is not the same as LCM.
Less heating of tissue with LMD versus LCM.
Laser Capture Microdissection
Emmert-Buck et al., 1996

7. X-Y and objective controls
The microscope
Microscope software
Laser power source
X-Y and objective controls
Microscope

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. A B C

10. Plant shoot tips – mature leaf and stem, young leaf and SAM
Harvest tissue ✓
Plant shoot tips – mature leaf and stem, young leaf and SAM
Fix/preserve tissue ✓
3:1 ethanol:acetic acid
Mount tissue on membrane
Leica PEN membrane slides
(Heat block)
Deparaffinise
Ethanol/ xylene; 100% ethanol; 95% ethanol; 70% ethanol
Stain material for identification
E.g. eosin, haematoxylin
Dissect tissue
Genomics Facility LMD 6000
Downstream processing
RNA, DNA, protein, carbohydrates, etc.

11. 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?

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), 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 (5th floor, Porter Hall).
Useful discussions were had with Dr Faik and with Loren Honaas from Penn. State.