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Journal of Plant Research

, Volume 123, Issue 6, pp 807–813 | Cite as

A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection

  • Hirokazu Takahashi
  • Hisae Kamakura
  • Yutaka Sato
  • Katsuhiro Shiono
  • Tomomi Abiko
  • Nobuhiro Tsutsumi
  • Yoshiaki Nagamura
  • Naoko K. Nishizawa
  • Mikio Nakazono
Technical Note

Abstract

Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4–6 days to about 5 h by using a recently developed microwave method; (2) the time of floating sections from ~10 min to less than 5 min, (3) the drying time from ~12 to 1 h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.

Keywords

Laser microdissection Microwave Paraffin-embedded section RNase inhibitor 

Notes

Acknowledgments

We thank Dr. Noriko Inada for assisting with the microwave preparation and for stimulating discussions. We thank Drs. Akira Endo, Eiji Nambara, Masayoshi Kawaguchi, Hikota Miyazawa, Saeko Konishi, Takeshi Izawa and Yuko Ogo for assistance in quantifying the RNA obtained from the LM-isolated samples, and Dr. Sin-ichi Arimura for stimulating discussions. This work was partly supported by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a grant from the Bio-oriented Technology Research Advancement Institution (Promotion of Basic Research Activities for Innovative Biosciences) and a grant from Ministry of Agriculture, Forestry and Fisheries of Japan.

References

  1. Asano T, Masumura T, Kusano H, Kikuchi S, Kurita A, Shimada H, Kadowaki K (2002) Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem. Plant J 32:401–408CrossRefPubMedGoogle Scholar
  2. Cai S, Lashbrook CC (2006) Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling. Plant J 48:628–637CrossRefPubMedGoogle Scholar
  3. Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA (1996) Laser capture microdissection. Science 274:998–1001CrossRefPubMedGoogle Scholar
  4. Emrich SJ, Barbazuk WB, Li L, Schnable PS (2007) Gene discovery and annotation using LCM-454 transcriptome sequencing. Genome Res 17:69–73CrossRefPubMedGoogle Scholar
  5. Goldsworthy SM, Stockton PS, Trempus CS, Foley JF, Maronpot RR (1999) Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol Carcinog 25:86–91CrossRefPubMedGoogle Scholar
  6. Hirano K, Aya K, Hobo T, Sakakibara H, Kojima M, Shim RA, Hasegawa Y, Ueguchi-Tanaka M, Matsuoka M (2008) Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice. Plant Cell Physiol 49:1429–1450CrossRefPubMedGoogle Scholar
  7. Hobo T, Suwabe K, Aya K, Suzuki G, Yano K, Ishimizu T, Fujita M, Kikuchi S, Hamada K, Miyano M, Fujioka T, Kaneko F, Kazama T, Mizuta Y, Takahashi H, Shiono K, Nakazono M, Tsutsumi N, Nagamura Y, Kurata N, Watanabe M, Matsuoka M (2008) Various spatiotemporal expression profiles of anther-expressed genes in rice. Plant Cell Physiol 49:1417–1428CrossRefPubMedGoogle Scholar
  8. Inada N, Wildermuth MC (2005) Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf. Planta 211:9–16CrossRefPubMedGoogle Scholar
  9. Jiao Y, Tausta SL, Gandotra N, Sun N, Liu T, Clay NK, Ceserani T, Chen M, Ma L, Holford M, Zhang HY, Zhao H, Deng XW, Nelson T (2009) A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies. Nat Genet 41:258–263CrossRefPubMedGoogle Scholar
  10. Kerk NM, Ceserani T, Tausta SL, Sussex IM, Nelson TM (2003) Laser capture microdissection of cells from plant tissues. Plant Physiol 132:27–35CrossRefPubMedGoogle Scholar
  11. Nakazono M, Qiu F, Borsuk LA, Schnable PS (2003) 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 15:583–596CrossRefPubMedGoogle Scholar
  12. Nelson T, Tausta SL, Gandotra N, Liu T (2006) Laser microdissection of plant tissue: what you see is what you get. Annu Rev Plant Biol 57:181–201CrossRefPubMedGoogle Scholar
  13. Ohtsu K, Takahashi H, Schnable PS, Nakazono M (2007) Cell type-specific gene expression profiling in plants by using a combination of laser microdissection and high-throughput technologies. Plant Cell Physiol 48:3–7CrossRefPubMedGoogle Scholar
  14. Schnable PS, Hochholdinger F, Nakazono M (2004) Global expression profiling applied to plant development. Curr Opin Plant Biol 7:50–56CrossRefPubMedGoogle Scholar
  15. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3CrossRefPubMedGoogle Scholar
  16. Suwabe K, Suzuki G, Takahashi H, Shiono K, Endo M, Yano K, Fujita M, Masuko H, Saito H, Fujioka T, Kaneko F, Kazama T, Mizuta Y, Kawagishi-Kobayashi M, Tsutsumi N, Kurata N, Nakazono M, Watanabe M (2008) Separated transcriptomes of male gametophyte and tapetum in rice: validity of a laser microdissection (LM) microarray. Plant Cell Physiol 49:1407–1416CrossRefPubMedGoogle Scholar
  17. Tang W, Coughlan S, Crane E, Beatty M, Duvick J (2006) The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola. Mol Plant Microbe Interact 19:1240–1250CrossRefPubMedGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer 2010

Authors and Affiliations

  • Hirokazu Takahashi
    • 1
  • Hisae Kamakura
    • 1
  • Yutaka Sato
    • 2
  • Katsuhiro Shiono
    • 1
  • Tomomi Abiko
    • 1
  • Nobuhiro Tsutsumi
    • 1
  • Yoshiaki Nagamura
    • 2
  • Naoko K. Nishizawa
    • 1
    • 3
  • Mikio Nakazono
    • 1
  1. 1.Graduate School of Agricultural and Life SciencesUniversity of TokyoTokyoJapan
  2. 2.National Institute of Agrobiological ScienceTsukubaJapan
  3. 3.Research Institute for Bioresources and BiotechnologyIshikawa Prefectural UniversityIshikawaJapan

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