Improved Method of RNA Isolation from Laser Capture Microdissection (LCM)-Derived Plant Tissues
Laser capture microdissection (LCM) is a tool to isolate desired and/or less accessible cells or tissues from a heterogeneous population. In the current method, we describe an efficient and cost-effective method to obtain both high-quality mRNA and miRNAs in sufficient quantity from LCM-derived plant tissues. The quality of the isolated RNA can be assessed using Bioanalyzer. Using modified stem-loop RT-PCR, we confirmed the presence of 21–24 nucleotide (nt) long mature miRNAs. This modified LCM-based method has been found to be suitable for the tissue-specific expression analysis of both genes and small RNAs (miRNAs).
Key wordsLCM Laser microdissection RNA isolation Gene expression miRNA Root meristem
V.G., A.S., and S.S. thank the Council for Scientific and Industrial Research (CSIR), India, and the National Institute of Plant Genome Research (NIPGR), New Delhi, India, for funding and internal grants. V.G. and A.S. also acknowledge the Department of Biotechnology (DBT), India, for fellowship. S.V. thanks the Department of Science and Technology-Science and Engineering Research Board (DST-SERB), India, for National Post Doctoral Fellowship (N-PDF). A.K.S. thanks NIPGR and DBT (Project Grant No. BT/PR12766/BPA/118/63/2015), New Delhi, India, for fellowship and grants. The authors declare no conflict of interests.
- 3.Birnbaum K, Jung JW, Wang JY, Lambert GM, Hirst JA, Galbraith DW, Benfey PN (2005) Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat Methods 2(8):615–619. https://doi.org/10.1038/nmeth0805-615CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Ohtsu K, Smith MB, Emrich SJ, Borsuk LA, Zhou R, Chen T, Zhang X, Timmermans MC, Beck J, Buckner B, Janick-Buckner D, Nettleton D, Scanlon MJ, Schnable PS (2007) Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.). Plant J 52(3):391–404. https://doi.org/10.1111/j.1365-313X.2007.03244.xCrossRefPubMedPubMedCentralGoogle Scholar
- 9.Brooks L 3rd, Strable J, Zhang X, Ohtsu K, Zhou R, Sarkar A, Hargreaves S, Elshire RJ, Eudy D, Pawlowska T, Ware D, Janick-Buckner D, Buckner B, Timmermans MC, Schnable PS, Nettleton D, Scanlon MJ (2009) Microdissection of shoot meristem functional domains. PLoS Genet 5(5):e1000476. https://doi.org/10.1371/journal.pgen.1000476CrossRefPubMedPubMedCentralGoogle Scholar