Skip to main content
SpringerLink
Log in

RNA Stable Isotope Probing (RNA-SIP)

  • Protocol
  • First Online:
Stable Isotope Probing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2046))

Abstract

Stable isotope probing is a combined molecular and isotopic technique used to probe the identity and function of uncultivated microorganisms within environmental samples. Employing stable isotopes of common elements such as carbon and nitrogen, RNA-SIP exploits an increase in the buoyant density of RNA caused by the active metabolism and incorporation of heavier mass isotopes into the RNA after cellular utilization of labeled substrates pulsed into the community. Labeled RNAs are subsequently separated from unlabeled RNAs by density gradient centrifugation followed by identification of the RNAs by sequencing. Therefore, RNA stable isotope probing is a culture-independent technique that provides simultaneous information about microbiome community, composition and function. This chapter presents the detailed protocol for performing an RNA-SIP experiment, including the formation, ultracentrifugation, and fractional analyses of stable isotope-labeled RNAs extracted from environmental samples.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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. Dumont MG, Murrell JC (2005) Innovation: stable isotope probing—linking microbial identity to function. Nat Rev Microbiol 3:499–504. https://doi.org/10.1038/nrmicro1162

    Article  CAS  PubMed  Google Scholar 

  2. Woese CR, Fox GE (1977) Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci U S A 74:5088–5090. https://doi.org/10.1073/PNAS.74.11.5088

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Amann RI, Ludwig WSK (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Whittenbury R, Phillips KC, Wilkinson JF (1970) Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61:205–218. https://doi.org/10.1099/00221287-61-2-205

    Article  CAS  PubMed  Google Scholar 

  5. Huber H, Thomm M, Königw H, Thies G, Stetter KO (1982) Methanococcus thermolithotrophicus, a novel thermophilic lithotrophic methanogen. Arch Microbiol 132:47–50. https://doi.org/10.1007/BF00690816

    Article  Google Scholar 

  6. Radajewski S, McDonald IR, Murrell JC (2003) Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms. Curr Opin Biotechnol 14:296–302. https://doi.org/10.1016/S0958-1669(03)00064-8

    Article  CAS  PubMed  Google Scholar 

  7. Hugenholtz P, Goebel BMPN (1998) Impact of culture independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180:4765–4774

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Radajewski S, Ineson P, Parekh NR, Murrell JC (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403:646–649. https://doi.org/10.1038/35001054

    Article  CAS  PubMed  Google Scholar 

  9. Neufeld JD, Dumont MG, Vohra J, Murrell JC (2007) Methodological considerations for the use of stable isotope probing in microbial ecology. Microb Ecol 53:435–442. https://doi.org/10.1007/s00248-006-9125-x

    Article  CAS  PubMed  Google Scholar 

  10. Boschker HTS, Nold SC, Wellsbury P, Bos D de GW, Pel R, Parkes RJCTE (1998) Direct linking of microbial populations to specific biogeochemical processes by C-13-labelling of biomarkers. Nature 392:801–805

    Article  CAS  Google Scholar 

  11. Whiteley AS, Manefield M, Lueders T (2006) Unlocking the ‘microbial black box’ using RNA-based stable isotope probing technologies. Curr Opin Biotechnol 17:67–71. https://doi.org/10.1016/J.COPBIO.2005.11.002

    Article  CAS  PubMed  Google Scholar 

  12. Whiteley AS, Thomson B, Lueders T, Manefield M (2007) RNA stable-isotope probing. Nat Protoc 2:838–844. https://doi.org/10.1038/nprot.2007.115

    Article  CAS  PubMed  Google Scholar 

  13. Huang WE, Ferguson A, Singer AC, Lawson K, Thompson IP, Kalin RM, Larkin MJ, Bailey MJ, Whiteley AS (2009) Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization. Appl Environ Microbiol 75:234–241. https://doi.org/10.1128/AEM.01861-08

    Article  CAS  PubMed  Google Scholar 

  14. Manefield M, Whiteley AS, Griffiths RI, Bailey MJ (2002) RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl Environ Microbiol 68:5367–5373. https://doi.org/10.1128/AEM.68.11.5367-5373.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ostle N, Whiteley AS, Bailey MJ, Sleep D, Ineson P, Manefield M (2003) Active microbial RNA turnover in a grassland soil estimated using a 13CO2 spike. Soil Biol Biochem 35:877–885. https://doi.org/10.1016/S0038-0717(03)00117-2

    Article  CAS  Google Scholar 

  16. Manefield M, Griffiths RI, Leigh MB, Fisher R, Whiteley AS (2005) Functional and compositional comparison of two activated sludge communities remediating coking effluent. Environ Microbiol 7:715–722. https://doi.org/10.1111/j.1462-2920.2004.00746.x

    Article  CAS  PubMed  Google Scholar 

  17. Schwarz A, Adetutu EM, Juhasz AL, Aburto-Medina A, Ball AS, Shahsavari E (2017) Microbial degradation of phenanthrene in pristine and contaminated sandy soil. Microb Ecol 75(4):888–902

    Article  Google Scholar 

  18. Liu P, Pommerenke B, Conrad R (2018) Identification of Syntrophobacteraceae as major acetate-degrading sulfate reducing bacteria in Italian paddy soil. Environ Microbiol 20:337–354. https://doi.org/10.1111/1462-2920.14001

    Article  CAS  PubMed  Google Scholar 

  19. Lueders T, Pommerenke B, Friedrich MW (2004) Stable-isotope probing of microorganisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil. Appl Environ Microbiol 70(10):5778–5786

    Article  CAS  Google Scholar 

  20. Lueders T, Manefield M, Friedrich MW (2003) Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients. Environ Microbiol 6:73–78. https://doi.org/10.1046/j.1462-2920.2003.00536.x

    Article  CAS  Google Scholar 

  21. Lueders T, Kindler R, Miltner A, Friedrich MW, Kaestner M (2006) Identification of bacterial micropredators distinctively active in a soil microbial food web. Appl Environ Microbiol 72:5342–5348

    Article  CAS  Google Scholar 

  22. Griffiths RI, Whiteley AS, O’Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol 66:5488–5491. https://doi.org/10.1128/AEM.66.12.5488-5491.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Murrel JC, Whiteley AS (2010) Stable isotope probing and related technologies. American Society for Microbiology Press, Washington, DC

    Google Scholar 

  24. Bradford LM, Vestergaard G, Táncsics A, Zhu B, Schloter M, Lueders T (2018) Transcriptome-stable isotope probing provides targeted functional and taxonomic insights into microaerobic pollutant-degrading aquifer microbiota. Front Microbiol 9:2696. https://doi.org/10.3389/fmicb.2018.02696

    Article  PubMed  PubMed Central  Google Scholar 

  25. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703. https://doi.org/10.1128/JB.173.2.697-703.1991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Stubner S (2002) Enumeration of 16S rDNA of desulfotomaculum lineage 1 in rice field soil by real-time PCR with SybrGreen™ detection. J Microbiol Methods 50:155–164. https://doi.org/10.1016/S0167-7012(02)00024-6

    Article  CAS  PubMed  Google Scholar 

  27. Lueders T, Wagner B, Claus P, Friedrich MW (2004) Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil. Environ Microbiol 6(1):60–72

    Article  CAS  Google Scholar 

  28. Manefield M, Whiteley AS, Ostle N, Ineson P, Bailey MJ (2002) Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function. Rapid Commun Mass Spectrom 16:2179–2183. https://doi.org/10.1002/rcm.782

    Article  CAS  PubMed  Google Scholar 

  29. Huang WE, Stoecker K, Griffiths R, Newbold L, Daims H, Whiteley AS, Wagner M (2007) Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ Microbiol 9:1878–1889. https://doi.org/10.1111/j.1462-2920.2007.01352.x

    Article  CAS  PubMed  Google Scholar 

  30. Read D, Huang WE, Whiteley AS (2015) Single cell microbial ecophysiology with Raman-FISH. Springer, Berlin, pp 65–76

    Google Scholar 

Download references

Acknowledgments

The authors wish to thank all the investigators over the years who have played a role in developing RNA-SIP as a method, especially professors Mike Manefield, Tillmann Lüeders, and Dr. Ian Douglas of Beckman Coulter. Particular thanks go to Professor Colin Murrell for stimulating discussions over the years on stable isotope enhanced microbial ecology. The Molecular Microbial Ecology Laboratory at UWA is funded by a range of sources, including the Australian Research Council Linkage Program (LP150101111 to A.S.W.), The Australia China Joint Research Centre (A.S.W., I.W.), a Chilean BECAS scholarship (B.M.G.), and a UWA IPRS scholarship (N.H.G.).

Author information

Authors and Affiliations

  1. Molecular Microbial Ecology Group, The UWA School of Agriculture and Enviornment (SAgE), The University of Western Australia, Crawley, WA, Australia

    Noor-Ul-Huda Ghori, Benjamin Moreira-Grez, Paton Vuong, Ian Waite, Tim Morald & Andrew S. Whiteley

  2. Department of Computer Science and Engineering, The University of Western Australia, Perth, WA, Australia

    Michael Wise

  3. Faculty of Science, The University of Western Australia, Crawley, WA, Australia

    Andrew S. Whiteley

Authors
  1. Noor-Ul-Huda Ghori
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Benjamin Moreira-Grez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Paton Vuong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ian Waite
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tim Morald
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Wise
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andrew S. Whiteley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrew S. Whiteley .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Southampton, Southampton, UK

    Marc G. Dumont

  2. School of Biological Sciences, University of Southampton, Southampton, UK

    Marcela Hernández García

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ghori, NUH. et al. (2019). RNA Stable Isotope Probing (RNA-SIP). In: Dumont, M., Hernández García, M. (eds) Stable Isotope Probing. Methods in Molecular Biology, vol 2046. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9721-3_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9721-3_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9720-6

  • Online ISBN: 978-1-4939-9721-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation