http://www.bio-protocol.org/e1417 Vol 5, Iss 5, Mar 05, 2015 1 Extraction of Small RNA and qPCR Validation of miRNAs in Vigna mungo Sujay Paul 1, 2 , Anirban Kundu 1 and Amita Pal 1* 1 Division of Plant Biology, Bose Institute, West Bengal, India; 2 Center for Genomic Sciences, National Autonomous University of Mexico, Morelos, Mexico *For correspondence: amita@jcbose.ac.in [Abstract] Small RNAs like microRNAs (miRNAs), small interfering RNAs (siRNAs) and other noncoding RNAs including snRNA and snoRNA have tremendous impact on eukaryotic gene regulation. Extraction of high quality small RNAs is an important prerequisite for experimental analyses of miRNAs. This will prevent RNA degradation and remove associated contaminations including polyphenols, polysaccharides and other secondary metabolites. In this protocol we describe a simple way to isolate small RNAs from the leaf tissues of Vigna mungo combining the protocols of two commercially available kits with some modifications. Materials and Reagents 1. Vigna mungo MYMIV-resistant recombinant inbred line, VMR84 2. mirPremier microRNA isolation kit (Sigma-Aldrich, catalog number: SNC10) 3. Mir-X miRNA FirstStrand synthesis and SYBR qRT-PCR kit (Takara Bio Company, Clontech, catalog number: 638314) 4. 2-mercaptoethanol (Sisco Research Laboratories, catalog number: 1324196) 5. Ethanol (Merck, catalog number: K41540783) 6. Liquid nitrogen and dry ice 7. RNase free water (Bangalore Genei, catalog number: 612151181001730) 8. Agarose (Sisco Research Laboratories, catalog number: 0140229) 9. Ethidium bromide (Sisco Research Laboratories, catalog number: 054817) 10. 1x TAE buffer (see Recipes) 11. Ethidium bromide stock (see Recipes) Equipment 1. Thermocycler (DNA Engine Cycler, model: PTC-200) 2. Real-time qPCR (Biorad iQ5 Real-Time PCR Detection System)