A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection
- 1.2k Downloads
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.
KeywordsLaser microdissection Microwave Paraffin-embedded section RNase inhibitor
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.
- 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
- 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
- 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
- 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