Skip to main content
SpringerLink
Log in

Guidelines for the Optimization and Validation of In Situ Hybridization

  • Protocol
  • First Online:
In Situ Hybridization Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2148))

Abstract

As RNA in situ hybridization (ISH) moves into the mainstream lab and increasingly into clinical adoption and additional multiplexing techniques are developed to enable further RNA ISH identification, a set of guidelines on the validation of ISH is required. These guidelines include choice of methods, appropriate controls, and protocol optimization as well as a central core message of understanding the target, understanding the ISH technique, and using the most appropriate controlling mechanisms to enable reproducible and trustworthy data to be obtained.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
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

Notes

  1. 1.

    ViewRNA is a product from Thermo Fisher Scientific.

  2. 2.

    RNAscope® is a registered trademark of Advanced Cell Diagnostics.

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. https://doi.org/10.1073/pnas.63.2.378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wojcik AM, Mosiolek M, Karcz J et al (2018) Whole mount in situ localization of miRNAs and mRNAs during somatic embryogenesis in Arabidopsis. Front Plant Sci 9:1277. https://doi.org/10.3389/fpls.2018.01277

    Article  PubMed  PubMed Central  Google Scholar 

  3. Koshiba-Takeuchi K (2018) Whole-mount and section in situ hybridization in mouse embryos for detecting mRNA expression and localization. Methods Mol Biol 1752:123–131. https://doi.org/10.1007/978-1-4939-7714-7_12

    Article  CAS  PubMed  Google Scholar 

  4. Richardson L, Stevenson P, Venkataraman S et al (2014) EMAGE: electronic mouse atlas of gene expression. Methods Mol Biol 1092:61–79. https://doi.org/10.1007/978-1-60327-292-6_5

    Article  CAS  PubMed  Google Scholar 

  5. Hochheiser H, Yanowitz J (2007) If I only had a brain: exploring mouse brain images in the Allen Brain Atlas. Biol Cell 99:403–409. https://doi.org/10.1042/BC20070031

    Article  PubMed  Google Scholar 

  6. Shen EH, Overly CC, Jones AR (2012) The Allen Human Brain Atlas: comprehensive gene expression mapping of the human brain. Trends Neurosci 35:711–714. https://doi.org/10.1016/j.tins.2012.09.005

    Article  CAS  PubMed  Google Scholar 

  7. Mahmood R, Mason I (2008) In-situ hybridization of radioactive riboprobes to RNA in tissue sections. Methods Mol Biol 461:675–686. https://doi.org/10.1007/978-1-60327-483-8_45

    Article  CAS  PubMed  Google Scholar 

  8. Pringle JH, Primrose L, Kind CN et al (1989) In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe. J Pathol 158:279–286. https://doi.org/10.1002/path.1711580403

    Article  CAS  PubMed  Google Scholar 

  9. Silahtaroglu AN, Tommerup N, Vissing H (2003) FISHing with locked nucleic acids (LNA): evaluation of different LNA/DNA mixmers. Mol Cell Probes 17:165–169

    Article  CAS  Google Scholar 

  10. Silahtaroglu AN, Nolting D, Dyrskjot L et al (2007) Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. Nat Protoc 2:2520–2528. https://doi.org/10.1038/nprot.2007.313

    Article  CAS  PubMed  Google Scholar 

  11. Turnock-Jones JJ, Le Quesne JP (2014) MicroRNA in situ hybridization in tissue microarrays. Methods Mol Biol 1211:85–93. https://doi.org/10.1007/978-1-4939-1459-3_8

    Article  CAS  PubMed  Google Scholar 

  12. Player AN, Shen LP, Kenny D et al (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J Histochem Cytochem 49:603–612. https://doi.org/10.1177/002215540104900507

    Article  CAS  PubMed  Google Scholar 

  13. Choi HMT, Schwarzkopf M, Fornace ME et al (2018) Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145. https://doi.org/10.1242/dev.165753

  14. Orjalo A Jr, Johansson HE, Ruth JL (2011) Stellarisâ„¢ fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection. Nat Methods 8:884. https://doi.org/10.1038/nmeth.f.349

    Article  CAS  Google Scholar 

  15. Kishi JY, Lapan SW, Beliveau BJ et al (2019) SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat Methods 16:533–544. https://doi.org/10.1038/s41592-019-0404-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Goltsev Y, Samusik N, Kennedy-Darling J et al (2018) Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell 174(968–981):e915. https://doi.org/10.1016/j.cell.2018.07.010

    Article  CAS  Google Scholar 

  17. Xia C, Babcock HP, Moffitt JR et al (2019) Multiplexed detection of RNA using MERFISH and branched DNA amplification. Sci Rep 9:7721. https://doi.org/10.1038/s41598-019-43943-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang X, Allen WE, Wright MA et al (2018) Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361. https://doi.org/10.1126/science.aat5691

  19. Wu C, Simonetti M, Rossell C et al (2018) RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples. Commun Biol 1:209. https://doi.org/10.1038/s42003-018-0218-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Humphries MP, McQuaid S, Craig SG et al (2019) Critical appraisal of programmed death ligand 1 reflex diagnostic testing: current standards and future opportunities. J Thorac Oncol 14:45–53. https://doi.org/10.1016/j.jtho.2018.09.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bingham V, Ong CW, James J et al (2016) PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry. Hum Pathol 47:95–103. https://doi.org/10.1016/j.humpath.2015.09.009

    Article  CAS  PubMed  Google Scholar 

  22. Ferrone CR, Ting DT, Shahid M et al (2016) The ability to diagnose intrahepatic cholangiocarcinoma definitively using novel branched DNA-enhanced albumin RNA in situ hybridization technology. Ann Surg Oncol 23:290–296. https://doi.org/10.1245/s10434-014-4247-8

    Article  PubMed  Google Scholar 

  23. Kang H, Antonarakis ES, Luo J et al (2018) Detection of AR-V7 transcript with RNA in situ hybridization in human salivary duct cancer. Oral Oncol 84:134–136. https://doi.org/10.1016/j.oraloncology.2018.06.026

    Article  CAS  PubMed  Google Scholar 

  24. Groelz D, Viertler C, Pabst D et al (2018) Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues. PLoS One 13:e0203608. https://doi.org/10.1371/journal.pone.0203608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Baena-Del Valle JA, Zheng Q, Hicks JL et al (2017) Rapid loss of RNA detection by in situ hybridization in stored tissue blocks and preservation by cold storage of unstained slides. Am J Clin Pathol 148:398–415. https://doi.org/10.1093/ajcp/aqx094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Warren M, Chung YJ, Howat WJ et al (2010) Irradiated Blm-deficient mice are a highly tumor prone model for analysis of a broad spectrum of hematologic malignancies. Leuk Res 34:210–220. https://doi.org/10.1016/j.leukres.2009.06.007

    Article  CAS  PubMed  Google Scholar 

  27. Evers DL, He J, Kim YH et al (2011) Paraffin embedding contributes to RNA aggregation, reduced RNA yield, and low RNA quality. J Mol Diagn 13:687–694. https://doi.org/10.1016/j.jmoldx.2011.06.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cronin M, Ghosh K, Sistare F et al (2004) Universal RNA reference materials for gene expression. Clin Chem 50:1464–1471. https://doi.org/10.1373/clinchem.2004.035675

    Article  CAS  PubMed  Google Scholar 

  29. Macabeo-Ong M, Shiboski CH, Silverman S et al (2003) Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression. Oral Oncol 39:638–647

    Article  CAS  Google Scholar 

  30. Chung JY, Braunschweig T, Williams R et al (2008) Factors in tissue handling and processing that impact RNA obtained from formalin-fixed, paraffin-embedded tissue. J Histochem Cytochem 56:1033–1042. https://doi.org/10.1369/jhc.2008.951863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Tongiorgi E, Righi M, Cattaneo A (1998) A non-radioactive in situ hybridization method that does not require RNase-free conditions. J Neurosci Methods 85:129–139

    Article  CAS  Google Scholar 

  32. von Ahlfen S, Missel A, Bendrat K et al (2007) Determinants of RNA quality from FFPE samples. PLoS One 2:e1261. https://doi.org/10.1371/journal.pone.0001261

    Article  CAS  Google Scholar 

  33. Schmeller J, Wessolly M, Mairinger E et al (2019) Setting out the frame conditions for feasible use of FFPE derived RNA. Pathol Res Pract 215:381–386. https://doi.org/10.1016/j.prp.2018.12.027

    Article  CAS  PubMed  Google Scholar 

  34. Zhang T, Tan P, Wang L et al (2017) RNALocate: a resource for RNA subcellular localizations. Nucleic Acids Res 45:D135–D138. https://doi.org/10.1093/nar/gkw728

    Article  CAS  PubMed  Google Scholar 

  35. Mas-Ponte D, Carlevaro-Fita J, Palumbo E et al (2017) LncATLAS database for subcellular localization of long noncoding RNAs. RNA 23:1080–1087. https://doi.org/10.1261/rna.060814.117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Griffiths-Jones S, Grocock RJ, van Dongen S et al (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 34:D140–D144. https://doi.org/10.1093/nar/gkj112

    Article  CAS  Google Scholar 

  37. Lennon G, Auffray C, Polymeropoulos M et al (1996) The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics 33:151–152. https://doi.org/10.1006/geno.1996.0177

    Article  CAS  PubMed  Google Scholar 

  38. Stenberg J, Nilsson M, Landegren U (2005) ProbeMaker: an extensible framework for design of sets of oligonucleotide probes. BMC Bioinformatics 6:229. https://doi.org/10.1186/1471-2105-6-229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gresner SM, Golanska E, Kulczycka-Wojdala D et al (2011) Selection of reference genes for gene expression studies in astrocytomas. Anal Biochem 408:163–165. https://doi.org/10.1016/j.ab.2010.09.010

    Article  CAS  PubMed  Google Scholar 

  40. Glare EM, Divjak M, Bailey MJ et al (2002) Beta-actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax 57:765–770. https://doi.org/10.1136/thorax.57.9.765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bingham V, McIlreavey L, Greene C et al (2017) RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples. Oncotarget 8:93392–93403. https://doi.org/10.18632/oncotarget.21851

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank members of the Histopathology/ISH Core Facility, CRUK-Cambridge Institute, and Nathan Benaich, Doug Winton, and Nikki March for kind permission to use images.

Author information

Authors and Affiliations

  1. Cancer Research UK, University of Cambridge, Cambridge, UK

    Julia Jones

  2. Abcam plc, Discovery Drive, Cambridge Biomedical Campus, Cambridge, UK

    William J. Howat

Authors
  1. Julia Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William J. Howat
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William J. Howat .

Editor information

Editors and Affiliations

  1. Molecular Histology, Bioneer A/S, Horsholm, Denmark

    Boye Schnack Nielsen

  2. Cancer Research UK, University of Cambridge, Cambridge, UK

    Julia Jones

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Jones, J., Howat, W.J. (2020). Guidelines for the Optimization and Validation of In Situ Hybridization. In: Nielsen, B.S., Jones, J. (eds) In Situ Hybridization Protocols . Methods in Molecular Biology, vol 2148. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0623-0_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0623-0_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0622-3

  • Online ISBN: 978-1-0716-0623-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation