Advertisement

Visualization of lncRNA by Single-Molecule Fluorescence In Situ Hybridization

  • Margaret Dunagin
  • Moran N. Cabili
  • John Rinn
  • Arjun Raj
Part of the Methods in Molecular Biology book series (MIMB, volume 1262)

Abstract

Single-molecule RNA fluorescence in situ hybridization is a technique that holds great potential for the study of long noncoding RNA. It enables quantification and spatial resolution of single RNA molecules within cells via hybridization of multiple, labeled nucleic acid probes to a target RNA. It has recently become apparent that single-molecule RNA FISH probes targeting noncoding RNA are more prone to off-target binding yielding spurious results than when targeting mRNA. Here we present a protocol for the application of single-molecule RNA FISH to the study of noncoding RNA as well as an experimental procedure for validating legitimate signals.

Key words

Single-molecule RNA FISH lncRNA Noncoding RNA Fluorescence in situ hybridization Single cell 

References

  1. 1.
    Ulitsky I, Bartel DP (2013) lincRNAs: genomics, evolution, and mechanisms. Cell 154:26–46PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Rinn JL, Chang HY (2012) Genome regulation by long noncoding RNAs. Annu Rev Biochem 81:145–166PubMedCrossRefGoogle Scholar
  3. 3.
    Kung JT, Colognori D, Lee JT (2013) Long noncoding RNAs: past, present, and future. Genetics 193:651–669PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Mercer TR, Dinger ME, Mattick JS (2009) Long non-coding RNAs: insights into functions. Nat Rev Genet 10:155–159PubMedCrossRefGoogle Scholar
  5. 5.
    Harrison PR, Conkie D, Paul J, Jones K (1973) Localisation of cellular globin messenger RNA by in situ hybridisation to complementary DNA. FEBS Lett 32:109–112PubMedCrossRefGoogle Scholar
  6. 6.
    Singer RH, Ward DC (1982) Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc Natl Acad Sci U S A 79:7331–7335PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Brown CJ, Hendrich BD, Rupert JL, Lafreniere RG, Xing Y, Lawrence J, Willard HF (1992) The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71:527–542PubMedCrossRefGoogle Scholar
  8. 8.
    Clemson CM, McNeil JA, Willard HF, Lawrence JB (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol 132:259–275PubMedCrossRefGoogle Scholar
  9. 9.
    Clemson CM, Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess A, Lawrence JB (2009) An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell 33:717–726PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A (2007) A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics 8:39PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Ip JY, Nakagawa S (2012) Long non-coding RNAs in nuclear bodies. Dev Growth Differ 54:44–54PubMedCrossRefGoogle Scholar
  12. 12.
    Sasaki YT, Ideue T, Sano M, Mituyama T, Hirose T (2009) MENepsilon/beta noncoding RNAs are essential for structural integrity of nuclear paraspeckles. Proc Natl Acad Sci U S A 106:2525–2530PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Sone M, Hayashi T, Tarui H, Agata K, Takeichi M, Nakagawa S (2007) The mRNA-like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons. J Cell Sci 120:2498–2506PubMedCrossRefGoogle Scholar
  14. 14.
    Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280:585–590PubMedCrossRefGoogle Scholar
  15. 15.
    Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5:877–879PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Bumgarner SL, Neuert G, Voight BF, Symbor-Nagrabska A, Grisafi P, van Oudenaarden A, Fink GR (2012) Single-cell analysis reveals that noncoding RNAs contribute to clonal heterogeneity by modulating transcription factor recruitment. Mol Cell 45:470–482PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Carpenter S, Aiello D, Atianand MK, Ricci EP, Gandhi P, Hall LL, Byron M, Monks B, Henry-Bezy M, Lawrence JB et al (2013) A long noncoding RNA mediates both activation and repression of immune response genes. Science 341:789–792PubMedCrossRefGoogle Scholar
  18. 18.
    Hacisuleyman E, Goff LA, Trapnell C, Williams A, Henao-Mejia J, Sun L, McClanahan P, Hendrickson DG, Sauvageau M, Kelley DR et al (2014) Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat Struct Mol Biol 21:198–206PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, Thomas K, Presser A, Bernstein BE, van Oudenaarden A et al (2009) Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A 106:11667–11672PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Kretz M, Siprashvili Z, Chu C, Webster DE, Zehnder A, Qu K, Lee CS, Flockhart RJ, Groff AF, Chow J et al (2013) Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493:231–235PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Maamar H, Cabili MN, Rinn J, Raj A (2013) linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis. Genes Dev 27:1260–1271PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Mohammad F, Pandey RR, Nagano T, Chakalova L, Mondal T, Fraser P, Kanduri C (2008) Kcnq1ot1/Lit1 noncoding RNA mediates transcriptional silencing by targeting to the perinucleolar region. Mol Cell Biol 28:3713–3728PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Wang KC, Yang YW, Liu B, Sanyal A, Corces-Zimmerman R, Chen Y, Lajoie BR, Protacio A, Flynn RA, Gupta RA et al (2011) A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472:120–124PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Raj A, Tyagi S (2010) Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 472:365–386PubMedCrossRefGoogle Scholar
  25. 25.
    Levesque MJ, Raj A (2013) Single chromosome transcriptional profiling reveals chromosome-level regulation of gene expression. Nat Methods 10:246. doi:10.1038/nmeth.2372 10.1038/nmeth.2372 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BioengineeringUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Broad Institute of Massachusetts Institute of Technology and HarvardCambridgeUSA
  3. 3.Department of Systems BiologyHarvard Medical SchoolBostonUSA
  4. 4.Department of Stem Cell and Regenerative BiologyHarvard UniversityCambridgeUSA

Personalised recommendations