Imaging individual microRNAs in single mammalian cells in situ

MicroRNAs (miRNAs) are potent negative regulators of gene expression that have been implicated in most major cellular processes. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, many fundamental questions still remain regarding miRNA function and their influence on cell cycle control. Considering recent reports on the impact of cell-to-cell fluctuations in gene expression on phenotypic diversity, it is likely that looking at the average miRNA expression of cell populations could result in the loss of important information connecting miRNA expression and cell function. Currently, however, there are no efficient techniques to quantify miRNA expression at the single-cell level. Here, a method is described for the detection of individual miRNA molecules in cancer cells using fluorescence in situ hybridization. The method combines the unique recognition properties of locked nucleic acid probes with enzyme-labeled fluorescence. Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots. In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.

[1]  T. Pederson,et al.  MicroRNA-206 colocalizes with ribosome-rich regions in both the nucleolus and cytoplasm of rat myogenic cells , 2006, Proceedings of the National Academy of Sciences.

[2]  Chang-Zheng Chen,et al.  MicroRNAs as oncogenes and tumor suppressors. , 2005, The New England journal of medicine.

[3]  V. Kim.  MicroRNA biogenesis: coordinated cropping and dicing , 2005, Nature Reviews Molecular Cell Biology.

[4]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[5]  Sonal Patel,et al.  A single-molecule method for the quantitation of microRNA gene expression , 2005, Nature Methods.

[6]  H. Horvitz,et al.  MicroRNA Expression in Zebrafish Embryonic Development , 2005, Science.

[7]  G. Dreyfuss,et al.  Numerous microRNPs in neuronal cells containing novel microRNAs. , 2003, RNA.

[8]  Jennifer Couzin.  A New Cancer Player Takes the Stage , 2005, Science.

[9]  M. Zavolan,et al.  miRNA in situ hybridization in formaldehyde and EDC–fixed tissues , 2009, Nature Methods.

[10]  C. Burge,et al.  Conserved Seed Pairing, Often Flanked by Adenosines, Indicates that Thousands of Human Genes are MicroRNA Targets , 2005, Cell.

[11]  H. Horvitz,et al.  MicroRNA expression profiles classify human cancers , 2005, Nature.

[12]  Jennifer Couzin.  Cancer biology. A new cancer player takes the stage. , 2005, Science.

[13]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

[14]  C. Croce,et al.  Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Gregory J. Hannon,et al.  MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies , 2005, Nature Cell Biology.

[16]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[17]  Anton J. Enright,et al.  Identification of Virus-Encoded MicroRNAs , 2004, Science.

[18]  Larisa M Haupt,et al.  Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR. , 2004, Nucleic acids research.

[19]  D. Tranchina,et al.  Stochastic mRNA Synthesis in Mammalian Cells , 2006, PLoS biology.

[20]  R. Haugland,et al.  The ELF-97 Alkaline Phosphatase Substrate Provides a Bright, Photostable, Fluorescent Signal Amplification Method for FISH , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[21]  Eugene Berezikov,et al.  Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification , 2007, Nature Protocols.

[22]  Scott A. Rifkin,et al.  Imaging individual mRNA molecules using multiple singly labeled probes , 2008, Nature Methods.

[23]  Wigard P Kloosterman,et al.  In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes , 2005, Nature Methods.

[24]  R. Plasterk,et al.  RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain. , 2005, RNA.

[25]  C. Croce,et al.  The role of microRNA genes in papillary thyroid carcinoma. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Mendell.  MicroRNAs: Critical Regulators of Development, Cellular Physiology and Malignancy , 2005, Cell cycle.

[27]  N. Rajewsky,et al.  A pancreatic islet-specific microRNA regulates insulin secretion , 2004, Nature.

[28]  F S Fay,et al.  Visualization of single RNA transcripts in situ. , 1998, Science.

[29]  M. Behlke,et al.  Design of LNA probes that improve mismatch discrimination , 2006, Nucleic acids research.

[30]  E. Lyon,et al.  Unlabeled oligonucleotide probes modified with locked nucleic acids for improved mismatch discrimination in genotyping by melting analysis. , 2005, BioTechniques.

[31]  Nóra Varga,et al.  Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. , 2004, Nucleic acids research.