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Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling

Nature Protocols volume 11, pages 2376–2388 (2016)Cite this article

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Abstract

This protocol enables transcriptome profiling of specific cell or tissue types that are isolated from tomato using laser microdissection (LM). To prepare tissue for LM, fruit samples are first fixed in optimal cutting temperature (OCT) medium and frozen in molds. The tissue is then sectioned using a cryostat before being dissected using an LM instrument. The RNAs contained in the harvested cells are purified and subjected to two rounds of amplification to yield sufficient quantities of RNA to generate cDNA libraries. Unlike several other techniques that are used to isolate specific cell types, LM has the advantage of being readily applied to any plant species without having to generate transgenic plants. Using the protocols described here, LM-mediated cell-type transcriptomic analysis of two samples requires ∼8 d from tissue harvest to RNA sequencing (RNA-seq), whereas each additional sample, up to a total of 12 samples, requires ∼1 additional day for the LM step. RNA obtained using this method has been successfully used for deep-coverage transcriptome profiling, which is a particularly effective strategy for identifying genes that are differentially expressed between cell or tissue types.

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Figure 1: Flowchart of laser capture microdissection for transcriptomic analysis.
Figure 2: Sample embedding in OCT medium.
Figure 3: Sample mounting using CryoJane tape transfer method on CFSA 1× slides.
Figure 4: Sample mounting on a frame slide membrane.
Figure 5: LM of fertilized ovaries.
Figure 6: Qualitative assessment of RNA integrity using a Fragment Analyzer.

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References

  1. Martin, L.B.B., Fei, Z., Giovannoni, J.J. & Rose, J.K.C. Catalyzing plant science research with RNA-seq. Front. Plant Sci. doi: http://dx.doi.org/10.3389/fpls.2013.00066 (2013).

  2. Hou, Z. et al. A cost effective RNA sequencing protocol for large-scale gene expression studies. Sci. Rep. 5, 9570 (2015).

    Article  Google Scholar 

  3. Rogers, E.D., Jackson, T., Moussaieff, A., Aharoni, A. & Benfey, P.N. Cell type-specific transcriptional profiling: implications for metabolite profiling. Plant J. 70, 5–17 (2012).

    Article  CAS  Google Scholar 

  4. Pattison, R.J. et al. Comprehensive tissue-specific transcriptome analysis reveals distinct regulatory programs during early tomato fruit development. Plant Physiol. 168, 1684–1701 (2015).

    Article  CAS  Google Scholar 

  5. Qiao, Z. & Libault, M. Unleashing the potential of the root hair cell as a single plant cell type model in root systems biology. Front. Plant Sci. 4, 484 (2013).

    Article  Google Scholar 

  6. Espina, V. et al. Laser-capture microdissection. Nat. Protoc. 1, 586–603 (2006).

    Article  CAS  Google Scholar 

  7. Birnbaum, K. et al. Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat. Methods 2, 615–619 (2005).

    Article  CAS  Google Scholar 

  8. Deal, R.B. & Henikoff, S. The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat. Protoc. 6, 56–68 (2011).

    Article  CAS  Google Scholar 

  9. Heiman, M., Kulicke, R., Fenster, R.J., Greengard, P. & Heintz, N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nat. Protoc. 9, 1282–1291 (2014).

    Article  CAS  Google Scholar 

  10. Brady, S.M. et al. A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318, 801–806 (2007).

    Article  CAS  Google Scholar 

  11. Bargmann, B.O.R. & Birnbaum, K.D. Fluorescence activated cell sorting of plant protoplasts. J. Vis. Exp. 18, 1673 (2010).

    Google Scholar 

  12. Petersson, S.V., Lindén, P., Moritz, T. & Ljung, K. Cell-type specific metabolic profiling of Arabidopsis thaliana protoplasts as a tool for plant systems biology. Metabolomics 11, 1679–1689 (2015).

    Article  CAS  Google Scholar 

  13. Gronlund, J.T., Eyres, A., Kumar, S., Buchanan-Wollaston, V. & Gifford, M.L. Cell specific analysis of Arabidopsis leaves using fluorescence activated cell sorting. J. Vis. Exp. 68, 4214 (2012).

    Google Scholar 

  14. Cheng, J. et al. Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308, 1149–1154 (2005).

    Article  CAS  Google Scholar 

  15. Barthelson, R.A., Lambert, G.M., Vanier, C., Lynch, R.M. & Galbraith, D.W. Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC Genomics 8, 340 (2007).

    Article  Google Scholar 

  16. Dinneny, J.R. et al. Cell identity mediates the response of Arabidopsis roots to abiotic stress. Science 320, 942–945 (2008).

    Article  CAS  Google Scholar 

  17. Chandran, D., Inada, N., Hather, G., Kleindt, C.K. & Wildermuth, M.C. Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators. Proc. Natl. Acad. Sci. USA 107, 460–465 (2010).

    Article  CAS  Google Scholar 

  18. Hacquart, S. et al. Laser capture microdissection of uredinia formed by Melampsora larici-populina revealed a transcriptional switch between biotrophy and sporulation. Mol. Plant Microbe Interact. 23, 1275–1286 (2010).

    Article  Google Scholar 

  19. Gaude, N., Bortfeld, S., Duensing, N., Lohse, M. & Krajinski, F. Arbuscule-containing and non-colonized cortical cells of mycorrhizal roots undergo extensive and specific reprogramming during arbuscular mycorrhizal development. Plant J. 69, 510–528 (2012).

    Article  CAS  Google Scholar 

  20. Hogekamp, C. & Küster, H. A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis. BMC Genomics 10.1186/1471-2164-14-306 doi: http://dx.doi.org/10.1186/1471-2164-14-306 (2013).

    Article  CAS  Google Scholar 

  21. Honaas, L.A. 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 Biol. 13 (2013).

    Article  CAS  Google Scholar 

  22. Nakazono, M., Qiu, F., Borsuk, L.A. & Schnable, P.S. Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 15, 583–596 (2003).

    Article  CAS  Google Scholar 

  23. Yu, P., Eggert, K., von Wirén, N., Li, C. & Hochholdinger, F. Cell type-specific gene expression analyses by RNA sequencing reveal local high nitrate-triggered lateral root initiation in shoot-borne roots of maize by modulating auxin-related cell cycle regulation. Plant Physiol. 169, 690–704 (2015).

    Article  CAS  Google Scholar 

  24. Matas, A.J., Agusti, J., Tadeo, F.R., Talon, M. & Rose, J.K.C. Tissue-specific transcriptome profiling of the citrus fruit epidermis and subepidermis using laser capture microdissection. J. Exp. Bot. 61, 3321–3330 (2010).

    Article  CAS  Google Scholar 

  25. Matas, A.J. et al. Tissue- and cell-type specific transcriptome profiling of expanding tomato fruit provides insights into metabolic and regulatory specialization and cuticle formation. Plant Cell 23, 3893–3910 (2011).

    Article  CAS  Google Scholar 

  26. Gao, C. et al. MicroRNA profiling analysis throughout tomato fruit development and ripening reveals potential regulatory role of RIN on microRNAs accumulation. Plant Biotechnol. J. 13, 370–382 (2015).

    Article  CAS  Google Scholar 

  27. Wang, Y. et al. Tomato nuclear proteome reveals the involvement of specific E2 ubiquitin-conjugating enzymes in fruit ripening. Genome Biol. 15, 548 (2014).

    Article  Google Scholar 

  28. Xu, J. et al. An extensive proteome map of tomato (Solanum lycopersicum) fruit pericarp. Proteomics 13, 3059–3063 (2013).

    CAS  PubMed  Google Scholar 

  29. Beisken, S. et al. Metabolic differences in ripening of Solanum lycopersicum 'Ailsa Craig' and three monogenic mutants. Sci. Data 1, 140029 (2014).

    Article  CAS  Google Scholar 

  30. Perez-Fons, L. et al. A genome-wide metabolomic resource for tomato fruit from. Sci. Rep. 4, 3859 (2014).

    Article  Google Scholar 

  31. Kerk, N.M., Ceserani, T., Tausta, S.L., Sussex, I.M. & Nelson, T.M. Laser capture microdissection of cells from plant tissues. Plant Physiol. 132, 27–35 (2003).

    Article  CAS  Google Scholar 

  32. Goldsworthy, S.M., Stockton, P.S., Trempus, C.S., Foley, J.F. & Maronpot, R.R. Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol. Carcinog. 25, 86–91 (1999).

    Article  CAS  Google Scholar 

  33. Inada, N. & Wildermuth, M.C. Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf. Planta 221, 9–16 (2005).

    Article  CAS  Google Scholar 

  34. Takahashi, H. et al. A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection. J. Plant Res. 123, 807–813 (2010).

    Article  CAS  Google Scholar 

  35. Cai, S. & Lashbrook, C.C. Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling. Plant J. 48, 628–637 (2006).

    Article  CAS  Google Scholar 

  36. Nelson, T., Tausta, S.L., Gandotra, N. & Liu, T. Laser microdissection of plant tissue: what you see is what you get. Annu. Rev. Plant Biol. 57, 181–201 (2006).

    Article  CAS  Google Scholar 

  37. Golubeva, Y.G., Smith, R.M. & Sternberg, L.R. Optimizing frozen sample preparation for laser microdissection: assessment of CryoJane tape-transfer system®. PLoS One 8, e66854 (2013).

    Article  CAS  Google Scholar 

  38. Vandewoestyne, M. et al. Laser capture microdissection: should an ultraviolet or infrared laser be used? Anal. Biochem. 439, 88–98 (2013).

    Article  CAS  Google Scholar 

  39. Brooks, L. III et al. Microdissection of shoot meristem functional domains. PLoS Genet. 5, e1000476 (2009).

    Article  Google Scholar 

  40. Clément-Ziza, M., Munnich, A., Lyonnet, S., Jaubert, F. & Besmond, C. Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions. RNA 14, 2698–2704 (2008).

    Article  Google Scholar 

  41. Suarez-Quian, A.A., Tirado, O.M., Munell, F. & Reventos, J. Laser capture microdissection to assess development. In Methods of Enzymology vol. 356, Laser Capture Microscopy and Microdissection (ed. Conn, P.M.) (Elsevier Science, 2002).

  42. Imbeaud, S. et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 33, 1–12 (2005).

    Article  Google Scholar 

  43. Schoor, O. et al. Moderate degradation does not preclude microarray analysis of small amounts of RNA. BioTechniques 35, 1192–1201 (2003).

    Article  CAS  Google Scholar 

  44. Gallego Romero, I., Pai, A.A., Tung, J. & Gilad, Y. RNA-seq: impact of RNA degradation on transcript quantification. BMC Biol. 12, 42 (2014).

    Article  Google Scholar 

  45. Ladanyi, A et al. Laser microdissection in translational and clinical research. Cytometry A 69A, 947–960 (2006).

    Article  CAS  Google Scholar 

  46. Nakamura, T. et al. Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection. Oncogene 23, 2385–2400 (2004).

    Article  CAS  Google Scholar 

  47. Turashvili, G. et al. Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis. BMC Cancer 7, 55 (2007).

    Article  Google Scholar 

  48. Kerman, I.A., Buck, B.J., Evans, S.J., Akil, H. & Watson, S.J. Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression. J. Neurosci. Methods 153, 71–85 (2006).

    Article  CAS  Google Scholar 

  49. Erickson, H.S. et al. Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples. Nat. Protoc. 4, 902–922 (2009).

    Article  CAS  Google Scholar 

  50. Kurimoto, K. & Saitou, M. Single-cell cDNA microarray profiling of complex biological processes of differentiation. Curr. Opin. Genet. Dev. 20, 470–477 (2010).

    Article  CAS  Google Scholar 

  51. Emrich, S.J., Barbazuk, W.B., Li, L. & Schnable, P.S. Gene discovery and annotation using LCM-454 transcriptome sequencing. Genome Res. 17, 69–73 (2007).

    Article  CAS  Google Scholar 

  52. Canas, R.A., Canales, J., Gomez-Maldonado, J., Avila, C. & Canovas, F.M. Transcriptome analysis in maritime pine using laser capture microdissection and 454 pyrosequencing. Tree Physiol. 34, 1278–1288 (2014).

    Article  CAS  Google Scholar 

  53. Schad, M., Lipton, M.S., Giavalisco, P., Smith, R.D. & Kehr, J. Evaluation of two-dimentional electrophoresis and liquid chromatography-tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples. Electrophoresis 26, 2729–2738 (2005).

    Article  CAS  Google Scholar 

  54. Schad, M., Mungur, R., Fiehn, O. & Kehr, J. Metabolic profiling of laser microdissected vascular bundles of Arabidopsis thaliana. Plant Methods 1, 2 (2005).

    Article  Google Scholar 

  55. Dembinsky, D. et al. Transcriptomic and proteomic analyses of pericycle cells of the maize primary root. Plant Physiol. 145, 575–588 (2007).

    Article  CAS  Google Scholar 

  56. Ruel, K. et al. Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana. New Phytol. 189, 99–113 (2009).

    Article  Google Scholar 

  57. Abbott, E., Hall, D., Hamberger, B. & Bohlmann, J. Laser microdissection of conifer stem tissues: isolation and analysis of high quality RNA, terpene synthase enzyme activity and terpenoid metabolites from resin ducts and cambial zone tissue of white spruce (Picea glauca). BMC Plant Biol. 10, 106 (2010).

    Article  Google Scholar 

  58. Falter, C., Ellinger, D., von Hulsen, B., Heim, R. & Voigt, C.A. Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis. Front. Plant Sci. doi: 10.3389/fpls.2015.00194 (2015).

  59. Farlik, M. et al. Single-cell DNA methylome sequencing and bioinformatics inference of epigenomic cell-state dynamics. Cell Rep. 10, 1386–1397 (2015).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge the Imaging Facility of Cornell University's Biotechnology Resource Center (Institute of Biotechnology) for the use of its Zeiss laser microdissection system. J.K.C.R. and C.C. were supported by a grant from the US National Science Foundation (Plant Genome Research Program; IOS-1339287), and Y.S. was partially supported by a Grant-in-Aid for JSPS Fellows from the Japan Society for the Promotion of Science (16J00582). A.J.M. was supported by the Spanish Ministry of Economy and Competitiveness with the Ramón y Cajal Fellowship (RYC-2011-08839).

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Author notes

  1. Laetitia B B Martin & Antonio J Matas

    Present address: Present addresses: Institut de Biologie Moléculaire des Plantes du CNRS, Strasbourg, France (L.B.B.M.) and Instituto de Hortofruticultura Subtropical y Mediterránea 'La Mayora' (IHSM-UMA-CSIC), Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain (A.J.M.).,

Authors and Affiliations

  1. Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York, USA

    Laetitia B B Martin, Antonio J Matas, Yoshihito Shinozaki, Carmen Catalá & Jocelyn K C Rose

  2. Boyce Thompson Institute for Plant Research, Ithaca, New York, USA

    Philippe Nicolas & Carmen Catalá

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Contributions

L.B.B.M., A.J.M., P.N. and Y.S. developed the protocol. L.B.B.M., P.N., C.C. and J.K.C.R. wrote the paper.

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Correspondence to Jocelyn K C Rose.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Annotation of cryostat features

a, block-holder; b, block in embedding mold; c, UV box; d, Object-holder; e, tightening screw; f, object-orientation knob; g, knife holder; h, angle control; i, anti-roll glass plate; j, CFSA 1X slide inside its protective wrapping; k, adhesive tape window; l, frame slide with PET-membrane; m, roller.

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Martin, L., Nicolas, P., Matas, A. et al. Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling. Nat Protoc 11, 2376–2388 (2016). https://doi.org/10.1038/nprot.2016.146

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