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Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments

Nature Protocols 3, 1395–1401 (2008) | Download Citation

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Abstract

We have developed protocols for rapidly quantifying the band intensities from nucleic acid chemical mapping gels at single-nucleotide resolution. These protocols are implemented in the software SAFA (semi-automated footprinting analysis) that can be downloaded without charge from http://safa.stanford.edu. The protocols implemented in SAFA have five steps: (i) lane identification, (ii) gel rectification, (iii) band assignment, (iv) model fitting and (v) band-intensity normalization. SAFA enables the rapid quantitation of gel images containing thousands of discrete bands, thereby eliminating a bottleneck to the analysis of chemical mapping experiments. An experienced user of the software can quantify a gel image in ∼20 min. Although SAFA was developed to analyze hydroxyl radical (·OH) footprints, it effectively quantifies the gel images obtained with other types of chemical mapping probes. We also present a series of tutorial movies that illustrate the best practices and different steps in the SAFA analysis as a supplement to this protocol.

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Acknowledgements

This work was funded by National Institutes of Health Grants P01-GM66275 to D.H., U54-GM072970 (National Centers for Biomedical Computation) to R.B.A., P41-EB0001979 to M.B., and K99/R00 (GM079953) award to A.L. and the NSF 0443508 for the RNA Ontology Consortium. Q.V. acknowledges the Howard Hughes Medical Institute for support.

Author information

Affiliations

  1. Department of Developmental Genetics and Bioinformatics, Wadsworth Center, Albany, New York 12208, USA.

    • Alain Laederach

  2. Department of Bioengineering, Stanford University, Stanford, California 94305, USA.

    • Alain Laederach
    •  & Russ B Altman

  3. Department of Genetics, Stanford University, Stanford, California 94305, USA.

    • Alain Laederach
    •  & Russ B Altman

  4. Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

    • Rhiju Das

  5. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.

    • Quentin Vicens

  6. Biomedical Informatics Program, Stanford University, Stanford, California 94304, USA.

    • Samuel M Pearlman

  7. Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

    • Michael Brenowitz

  8. Department of Biochemistry, Stanford University, Stanford, California 94305, USA.

    • Daniel Herschlag

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Correspondence to Alain Laederach.

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DOI

https://doi.org/10.1038/nprot.2008.134

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