Skip to main content
SpringerLink
Log in

Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as

  • Protocol
  • First Online:
Circular RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1724))

Abstract

Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA. The protocol can be adapted to circRNAs that coexist with overlapping, noncircular mRNA isoforms produced from the same genetic locus.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 69.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 89.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 119.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gall JG, Pardue ML (1969) Formation and detection of RNA–DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63:378–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. John HA, Birnstiel ML, Jones KW (1969) RNA-DNA hybrids at the cytological level. Nature 223:582–587

    Article  CAS  PubMed  Google Scholar 

  3. 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 79:7331–7335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lawrence JB, Singer RH (1986) Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45:407–415

    Article  CAS  PubMed  Google Scholar 

  5. Femino AM (1998) Visualization of single RNA transcripts in situ. Science 280:585–590. https://doi.org/10.1126/science.280.5363.585

    Article  CAS  PubMed  Google Scholar 

  6. Raj A, van den Bogaard P, Rifkin SA et al (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5:877–879. https://doi.org/10.1038/nmeth.1253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Raj A, Tyagi S (2010) Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol 472:365–386. https://doi.org/10.1016/S0076-6879(10)72004-8

    Article  CAS  PubMed  Google Scholar 

  8. Batish M, van den Bogaard P, Kramer FR, Tyagi S (2012) Neuronal mRNAs travel singly into dendrites. Proc Natl Acad Sci U S A 109:4645–4650. https://doi.org/10.1073/pnas.1111226109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  10. Trcek T, Chao JA, Larson DR et al (2012) Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat Protoc 7:408–419. https://doi.org/10.1038/nprot.2011.451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging technology. Nat Methods 8:S12–S19. https://doi.org/10.1038/nmeth.1573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gaspar I, Ephrussi A (2015) Strength in numbers: quantitative single-molecule RNA detection assays. WIREs Dev Biol 4:135–150. https://doi.org/10.1002/wdev.170

    Article  CAS  Google Scholar 

  13. Lubeck E, Cai L (2012) Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat Methods 9:743–748. https://doi.org/10.1038/nmeth.2069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Vargas DY, Raj A, Marras SAE et al (2005) Mechanism of mRNA transport in the nucleus. Proc Natl Acad Sci U S A 102:17008–17013. https://doi.org/10.1073/pnas.0505580102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Schwanhäusser B, Busse D, Li N et al (2011) Global quantification of mammalian gene expression control. Nature 473:337–342. https://doi.org/10.1038/nature10098

    Article  PubMed  Google Scholar 

  16. Femino A, Fogarty K, Lifshitz LM et al (2003) Visualization of single molecules of mRNA in situ. Methods Enzymol 361:245–304. https://doi.org/10.1016/S0076-6879(03)61015-3

    Article  CAS  PubMed  Google Scholar 

  17. Shaw G, Morse S, Ararat M, Graham FL (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J 16:869–871. https://doi.org/10.1096/fj.01-0995fje

    CAS  PubMed  Google Scholar 

  18. Lin Y-C, Boone M, Meuris L et al (2014) Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat Commun 5:4767. https://doi.org/10.1038/ncomms5767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nakayama Y, Wada A, Inoue R et al (2014) A rapid and efficient method for neuronal induction of the P19 embryonic carcinoma cell line. J Neurosci Methods 227:100–106. https://doi.org/10.1016/j.jneumeth.2014.02.011

    Article  CAS  PubMed  Google Scholar 

  20. Benesh RE, Benesh R (1953) Enzymatic removal of oxygen for polarography and related methods. Science 118:447–448

    Article  Google Scholar 

  21. Ha T, Tinnefeld P (2012) Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu Rev Phys Chem 63:595–617. https://doi.org/10.1146/annurev-physchem-032210-103340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Milo R, Jorgensen P, Moran U et al (2010) BioNumbers—the database of key numbers in molecular and cell biology. Nucleic Acids Res 38:D750–D753. https://doi.org/10.1093/nar/gkp889

    Article  CAS  PubMed  Google Scholar 

  23. Phillips R, Kondev J, Theriot J, Garcia H (2012) Physical biology of the cell, 2nd edn. Garland Science, London and New York

    Google Scholar 

  24. Milo R, Phillips R (2015) Cell biology by the numbers. Garland Science, London and New York

    Google Scholar 

  25. Jambor H, Brunel C, Ephrussi A (2011) Dimerization of oskar 3’ UTRs promotes hitchhiking for RNA localization in the Drosophila oocyte. RNA 17:2049–2057. https://doi.org/10.1261/rna.2686411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Park HY, Lim H, Yoon YJ et al (2014) Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science 343:422–424. https://doi.org/10.1126/science.1239200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Buxbaum AR, Wu B, Singer RH (2014) Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science 343:419–422. https://doi.org/10.1126/science.1242939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Akbalik G, Schuman EM (2014) Molecular biology. mRNA, live and unmasked. Science 343:375–376. https://doi.org/10.1126/science.1249623

    Article  CAS  PubMed  Google Scholar 

  29. Buxbaum AR, Yoon YJ, Singer RH, Park HY (2015) Single-molecule insights into mRNA dynamics in neurons. Trends Cell Biol 25:468–475. https://doi.org/10.1016/j.tcb.2015.05.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cabili MN, Dunagin MC, McClanahan PD et al (2015) Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biol 16:20. https://doi.org/10.1038/nmeth.2589

    Article  PubMed  PubMed Central  Google Scholar 

  31. Dunagin M, Cabili MN, Rinn J, Raj A (2014) Methods Mol Biol 1262:3–19. https://doi.org/10.1007/978-1-4939-2253-6_1

    Article  Google Scholar 

  32. Memczak S, Jens M, Elefsinioti A et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338. https://doi.org/10.1038/nature11928

    Article  CAS  PubMed  Google Scholar 

  33. Gaspar I, Wippich F, Ephrussi A (2017) Enzymatic production of single molecule FISH and RNA capture probes. RNA 23(10):1582–1591. https://doi.org/10.1261/rna.061184.117

    Article  PubMed  PubMed Central  Google Scholar 

  34. Salzman J (2016) Circular RNA expression: its potential regulation and function. Trends Genet 32:309–316. https://doi.org/10.1016/j.tig.2016.03.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ebbesen KK, Hansen TB, Kjems J (2016) Insights into circular RNA biology. RNA Biol:1–11. https://doi.org/10.1080/15476286.2016.1271524

  36. Salzman J, Gawad C, Wang PL et al (2012) Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One 7:e30733. https://doi.org/10.1371/journal.pone.0030733

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jeck WR, Sorrentino JA, Wang K et al (2013) Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19:141–157. https://doi.org/10.1261/rna.035667.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ashwal-Fluss R, Meyer M, Pamudurti NR et al (2014) circRNA biogenesis competes with pre-mRNA splicing. Mol Cell 56:55–66. https://doi.org/10.1016/j.molcel.2014.08.019

    Article  CAS  PubMed  Google Scholar 

  39. Zhang X-O, Wang H-B, Zhang Y et al (2014) Complementary sequence-mediated exon circularization. Cell 159:134–147. https://doi.org/10.1016/j.cell.2014.09.001

    Article  CAS  PubMed  Google Scholar 

  40. Starke S, Jost I, Rossbach O et al (2015) Exon circularization requires canonical splice signals. Cell Rep 10:103–111. https://doi.org/10.1016/j.celrep.2014.12.002

    Article  CAS  PubMed  Google Scholar 

  41. Rybak-Wolf A, Stottmeister C, Glažar P et al (2015) Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed. Mol Cell 58:870–885. https://doi.org/10.1016/j.molcel.2015.03.027

    Article  CAS  PubMed  Google Scholar 

  42. Hansen TB, Wiklund ED, Bramsen JB et al (2011) miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. EMBO J 30:4414–4422. https://doi.org/10.1038/emboj.2011.359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hansen TB, Jensen TI, Clausen BH et al (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384–388. https://doi.org/10.1038/nature11993

    Article  CAS  PubMed  Google Scholar 

  44. Bahar Halpern K, Caspi I, Lemze D et al (2015) Nuclear retention of mRNA in mammalian tissues. Cell Rep 13:2653–2662. https://doi.org/10.1016/j.celrep.2015.11.036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bahar Halpern K, Itzkovitz S (2016) Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues. Methods 98:134–142. https://doi.org/10.1016/j.ymeth.2015.11.015

    Article  CAS  PubMed  Google Scholar 

  46. Vargas DY, Shah K, Batish M et al (2011) Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell 147:1054–1065. https://doi.org/10.1016/j.cell.2011.10.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Orjalo AV, Johansson HE (2016) Stellaris® RNA fluorescence in situ hybridization for the simultaneous detection of immature and mature long noncoding RNAs in adherent cells. Methods Mol Biol 1402:119–134. https://doi.org/10.1007/978-1-4939-3378-5_10

    Article  CAS  PubMed  Google Scholar 

  48. Jeck WR, Sharpless NE (2014) Detecting and characterizing circular RNAs. Nat Biotechnol 32:453–461. https://doi.org/10.1038/nbt.2890

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lyubimova A, Itzkovitz S, Junker JP et al (2013) Single-molecule mRNA detection and counting in mammalian tissue. Nat Protoc 8:1743–1758. https://doi.org/10.1038/nprot.2013.109

    Article  PubMed  Google Scholar 

  50. Halpern KB, Tanami S, Landen S et al (2015) Bursty gene expression in the intact mammalian liver. Mol Cell 58:147–156. https://doi.org/10.1016/j.molcel.2015.01.027

    Article  PubMed Central  Google Scholar 

  51. Ben-Moshe S, Itzkovitz S (2016) Bursting through the cell cycle. Elife 5:e14953. https://doi.org/10.7554/eLife.14953

    Article  PubMed  PubMed Central  Google Scholar 

  52. Mortazavi A, Williams BA, McCue K et al (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628. https://doi.org/10.1038/nmeth.1226

    Article  CAS  PubMed  Google Scholar 

  53. Kellis M, Wold B, Snyder MP et al (2014) Defining functional DNA elements in the human genome. Proc Natl Acad Sci U S A 111:6131–6138. https://doi.org/10.1073/pnas.1318948111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Coassin SR, Orjalo AV, Semaan SJ, Johansson HE (2014) Simultaneous detection of nuclear and cytoplasmic RNA variants utilizing Stellaris® RNA fluorescence in situ hybridization in adherent cells. Methods Mol Biol 1211:189–199. https://doi.org/10.1007/978-1-4939-1459-3_15

    Article  CAS  PubMed  Google Scholar 

  55. Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. Methods Mol Biol 714:3–13. https://doi.org/10.1007/978-1-61779-005-8_1

    Article  CAS  PubMed  Google Scholar 

  56. Shi X, Lim J, Ha T (2010) Acidification of the oxygen scavenging system in single-molecule fluorescence studies: in situ sensing with a ratiometric dual-emission probe. Anal Chem 82:6132–6138. https://doi.org/10.1021/ac1008749

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Rasnik I, McKinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893. https://doi.org/10.1038/nmeth934

    Article  CAS  PubMed  Google Scholar 

  58. Cordes T, Vogelsang J, Tinnefeld P (2009) On the mechanism of Trolox as antiblinking and antibleaching reagent. J Am Chem Soc 131:5018–5019. https://doi.org/10.1021/ja809117z

    Article  CAS  PubMed  Google Scholar 

  59. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Mueller F, Senecal A, Tantale K et al (2013) FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10:277–278. https://doi.org/10.1038/nmeth.2406

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft [RA838/5-1 to N.R.] and the Berlin Institute of Health [CRG2aTP7 to N. R.]. We thank Arjun Raj and our BIMSB colleagues Alexander Loewer, Dhana Friedrich, Stefan Preibisch, and Marcel Schilling for discussions and advice.

Author information

Authors and Affiliations

  1. Systems Biology of Gene-Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center (MDC) for Molecular Medicine in the Helmholtz Association, Berlin, Germany

    Christine Kocks, Anastasiya Boltengagen, Monika Piwecka, Agnieszka Rybak-Wolf & Nikolaus Rajewsky

Authors
  1. Christine Kocks
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anastasiya Boltengagen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Monika Piwecka
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Agnieszka Rybak-Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nikolaus Rajewsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christine Kocks .

Editor information

Editors and Affiliations

  1. Department of Internal Medicine III Klaus Tschira Institute for Integrative Computational Cardiology, University Hospital Heidelberg, Heidelberg, Germany

    Christoph Dieterich

  2. Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

    Argyris Papantonis

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kocks, C., Boltengagen, A., Piwecka, M., Rybak-Wolf, A., Rajewsky, N. (2018). Single-Molecule Fluorescence In Situ Hybridization (FISH) of Circular RNA CDR1as. In: Dieterich, C., Papantonis, A. (eds) Circular RNAs. Methods in Molecular Biology, vol 1724. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7562-4_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7562-4_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7561-7

  • Online ISBN: 978-1-4939-7562-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation