title: An Efficient LCM-Based Method for Tissue Specific Expression Analysis of Genes and miRNAs.
abstract: Laser Capture Microdissection (LCM) is a powerful tool to isolate and study gene expression pattern of desired and less accessible cells or tissues from a heterogeneous population. Existing LCM-based methods fail to obtain high quality RNA including small RNAs from small microdissected plant tissue and therefore, are not suitable for miRNA expression studies. Here, we describe an efficient and cost-effective method to obtain both high quality RNA and miRNAs from LCM-derived embryonic root apical meristematic tissue, which is difficult to access. We have significantly modified and improved the tissue fixation, processing, sectioning and RNA isolation steps and minimized the use of kits. Isolated RNA was checked for quality with bioanalyzer and used for gene expression studies. We have confirmed the presence of 19-24 nucleotide long mature miRNAs using modified stem-loop RT-PCR. This modified LCM-based method is suitable for tissue specific expression analysis of both genes and small RNAs (miRNAs).
authors: National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110067, India.
authors: Vibhav
authors: V
authors: Gautam
authors: National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110067, India.
authors: Archita
authors: A
authors: Singh
authors: National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110067, India.
authors: Sharmila
authors: S
authors: Singh
authors: National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110067, India.
authors: Ananda K
authors: AK
authors: Sarkar
mesh_terms: D017360:Arabidopsis / Q000196:embryology / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D061346:Laser Capture Microdissection / Q000379:methods*; D018519:Meristem / Q000166:cytology / Q000235:genetics*; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008867:Microtomy / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger