Protocol | Published:

Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR

Nature Protocols volume 10, pages 11981211 (2015) | Download Citation

Abstract

Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Gene Expression Omnibus

References

  1. 1.

    , & Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006).

  2. 2.

    , , , & Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. Biol. Proced. Online 3, 19–25 (2001).

  3. 3.

    , , & Defining transcribed regions using RNA-seq. Nat. Protoc. 5, 255–266 (2010).

  4. 4.

    et al. A concise guide to cDNA microarray analysis. BioTechniques 29, 548–550, 552–554, 556 passim (2000).

  5. 5.

    & Identification of newly transcribed RNA. Curr. Protoc. Mol. Biol. 10, 4.10.1–4.10.12 (2007).

  6. 6.

    & Isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Nat. Protoc. 1, 3094–3100 (2006).

  7. 7.

    Using nuclear run-on transcription assays in RNAi studies. Methods Mol. Biol. 744, 199–209 (2011).

  8. 8.

    Nuclear run-on assay. Cold Spring Harb. Protoc. 2009 (2009).

  9. 9.

    et al. Novel DNA microarray system for analysis of nascent mRNAs. DNA Res. 15, 241–251 (2008).

  10. 10.

    , & Mobilization-competent lentiviral vector-mediated sustained transcriptional modulation of HIV-1 expression. Mol. Ther. 17, 360–368 (2009).

  11. 11.

    et al. Nuclear run-on assay using biotin labeling, magnetic bead capture and analysis by fluorescence-based RT-PCR. BioTechniques 29, 1012–1014, 1016–1017 (2000).

  12. 12.

    , & Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

  13. 13.

    , , , & MYC regulates the non-coding transcriptome. Oncotarget 5, 12543–12554 (2015).

  14. 14.

    et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).

  15. 15.

    et al. Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs. Proc. Natl. Acad. Sci. USA 112, E297–E302 (2015).

  16. 16.

    et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010).

  17. 17.

    et al. A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol. 8, e1000384 (2010).

  18. 18.

    et al. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev. 25, 742–754 (2011).

  19. 19.

    et al. Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells. Mol. Cell 50, 212–222 (2013).

  20. 20.

    et al. A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells. Cell 145, 622–634 (2011).

  21. 21.

    , , , & Small RNA-mediated epigenetic myostatin silencing. Mol. Ther. Nucleic Acids 1, e23 (2012).

  22. 22.

    , , , & Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells. Nucleic Acids Res. 37, 2984–2995 (2009).

  23. 23.

    , , & Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305, 1289–1292 (2004).

  24. 24.

    & Inhibiting transcription of chromosomal DNA using antigene RNAs. Nucleic Acids Symp. Ser. (Oxf.) 367–368 (2005).

  25. 25.

    , & Perspectives on the mechanism of transcriptional regulation by long non-coding RNAs. Epigenetics 9 (2013).

  26. 26.

    & Quantitative RT-PCR on CYP1A1 heterogeneous nuclear RNA: a surrogate for the in vitro transcription run-on assay. BioTechniques 20, 470–477 (1996).

  27. 27.

    , , & Specific and potent RNAi in the nucleus of human cells. Nat. Struct. Mol. Biol. 12, 133–137 (2005).

  28. 28.

    , , , & RNAi factors are present and active in human cell nuclei. Cell Rep. 6, 211–221 (2014).

  29. 29.

    The MicroRNA biology of the mammalian nucleus. Mol. Ther. Nucleic Acids 3, e188 (2014).

  30. 30.

    , , & Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).

  31. 31.

    et al. Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Res. 22, 947–956 (2012).

  32. 32.

    & Genome-wide technology for determining RNA stability in mammalian cells. RNA Biol. 9, 1233–1238 (2012).

  33. 33.

    et al. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14, 1959–1972 (2008).

  34. 34.

    & Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. USA 105, 15779–15784 (2008).

  35. 35.

    et al. Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA. Methods 67, 45–54 (2014).

  36. 36.

    et al. Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response. Proc. Natl. Acad. Sci. USA 110, 2240–2245 (2013).

  37. 37.

    et al. BRIC-seq: A genome-wide approach for determining RNA stability in mammalian cells. Methods 67, 55–63 (2014).

  38. 38.

    et al. Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. Mol. Biol. Cell 10, 211–223 (1999).

  39. 39.

    , , , & Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP). BioTechniques 22, 308–312 (1997).

  40. 40.

    et al. Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells. Chromosoma 108, 325–335 (1999).

  41. 41.

    , , & Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles. J. Cell Biol. 146, 543–558 (1999).

  42. 42.

    et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034 (2002).

  43. 43.

    et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009).

  44. 44.

    , , , & A practical approach to RT-qPCR: publishing data that conform to the MIQE guidelines. Methods 50, S1–5 (2010).

  45. 45.

    & Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408 (2001).

  46. 46.

    A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).

  47. 47.

    & Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

  48. 48.

    et al. Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells. Cell Biol. Int. 34, 663–668 (2010).

  49. 49.

    & Western blot: technique, theory, and trouble shooting. N. Am. J. Med. Sci. 4, 429–434 (2012).

  50. 50.

    et al. Control of cell growth by c-Myc in the absence of cell division. Curr. Biol. 9, 1255–1258 (1999).

  51. 51.

    , & Requirement for pre-existing of p21 to prevent doxorubicin-induced apoptosis through inhibition of caspase-3 activation. Mol. Cell. Biochem. 291, 139–144 (2006).

  52. 52.

    et al. Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program. Genes Dev. 20, 601–612 (2006).

Download references

Acknowledgements

T.C.R. is supported by a Medical Research Council UK Centenary Early Career Award. This is The Scripps Research Institute manuscript no. 29005.

Author information

Affiliations

  1. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA.

    • Thomas C Roberts
    • , Jonathan R Hart
    • , Marc S Weinberg
    • , Peter K Vogt
    •  & Kevin V Morris
  2. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

    • Thomas C Roberts
  3. Sanford-Burnham Medical Research Institute, Development, Aging and Regeneration Program, La Jolla, California, USA.

    • Thomas C Roberts
  4. Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland.

    • Minna U Kaikkonen
  5. Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa.

    • Marc S Weinberg
  6. HIV Pathogenesis Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa.

    • Marc S Weinberg
  7. School of Biotechnology and Biomedical Sciences, University of New South Wales, Sydney, New South Wales, Australia.

    • Kevin V Morris

Authors

  1. Search for Thomas C Roberts in:

  2. Search for Jonathan R Hart in:

  3. Search for Minna U Kaikkonen in:

  4. Search for Marc S Weinberg in:

  5. Search for Peter K Vogt in:

  6. Search for Kevin V Morris in:

Contributions

T.C.R., J.R.H., M.U.K., M.S.W., P.K.V. and K.V.M. contributed to the protocol and analyzed data. Experimental work was performed by T.C.R. and J.R.H. All authors contributed to the protocol and analyzed data. T.C.R. wrote the initial draft and all authors contributed to the final draft.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Thomas C Roberts.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Table 1, Supplementary Results, Supplementary Discussion and Supplementary Methods

Excel files

  1. 1.

    Supplementary Data

    Additional validation data for NRO-RT-qPCR reference genes.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2015.076

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.