Biomaterial Database

In situ localization of small RNAs in plants by using LNA probes. 2012

Marie Javelle, and Marja C P Timmermans

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

Small RNAs have crucial roles in numerous aspects of plant biology. Despite our current understanding of their biogenesis and mechanisms of action, the biological function of small RNAs, particularly miRNAs, remains largely unknown. To decipher small RNA function, knowledge about their spatiotemporal patterns of expression is essential. Here we report an in situ hybridization method for the precise localization of small RNAs in plants by using locked nucleic acid (LNA) oligonucleotide probes. This method has been adapted from protocols used to detect messenger RNAs in formaldehyde-fixed and paraffin-embedded tissue sections, but it includes essential optimizations in key prehybridization, hybridization and posthybridization steps. Most importantly, optimization of probe concentration and hybridization temperature is required for each unique LNA probe. We present the detailed protocol starting from sectioned tissues, and we include troubleshooting tips and recommended controls. This method has been used successfully in several plant species and can be completed within 2-6 d.

UI	MeSH Term	Description	Entries
D009841	Oligonucleotides	Polymers made up of a few (2-20) nucleotides. In molecular genetics, they refer to a short sequence synthesized to match a region where a mutation is known to occur, and then used as a probe (OLIGONUCLEOTIDE PROBES). (Dorland, 28th ed)	Oligonucleotide
D010944	Plants	Multicellular, eukaryotic life forms of kingdom Plantae. Plants acquired chloroplasts by direct endosymbiosis of CYANOBACTERIA. They are characterized by a mainly photosynthetic mode of nutrition; essentially unlimited growth at localized regions of cell divisions (MERISTEMS); cellulose within cells providing rigidity; the absence of organs of locomotion; absence of nervous and sensory systems; and an alternation of haploid and diploid generations. It is a non-taxonomical term most often referring to LAND PLANTS. In broad sense it includes RHODOPHYTA and GLAUCOPHYTA along with VIRIDIPLANTAE.	Plant
D015341	Nucleic Acid Probes	Nucleic acid which complements a specific mRNA or DNA molecule, or fragment thereof; used for hybridization studies in order to identify microorganisms and for genetic studies.	Acid Probes, Nucleic,Probes, Nucleic Acid
D017403	In Situ Hybridization	A technique that localizes specific nucleic acid sequences within intact chromosomes, eukaryotic cells, or bacterial cells through the use of specific nucleic acid-labeled probes.	Hybridization in Situ,Hybridization, In Situ,Hybridizations, In Situ,In Situ Hybridizations
D058727	RNA, Small Untranslated	Short RNA, about 200 base pairs in length or shorter, that does not code for protein.	Short Noncoding RNA,Small Non-Coding RNA,Small Non-Messenger RNA,Small Non-Protein-Coding RNA,Small Noncoding RNA,Small Untranslated RNA,sncRNA,sncRNAs,Non-Coding RNA, Small,Non-Messenger RNA, Small,Non-Protein-Coding RNA, Small,Noncoding RNA, Short,Noncoding RNA, Small,RNA, Short Noncoding,RNA, Small Non-Coding,RNA, Small Non-Messenger,RNA, Small Non-Protein-Coding,RNA, Small Noncoding,Small Non Coding RNA,Small Non Messenger RNA,Small Non Protein Coding RNA,Untranslated RNA, Small