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

Nature Protocols volume 3, pages 1395–1401 (2008)Cite this article

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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Figure 1: Basic premise of the SAFA software.
Figure 2: Basic principles of gel rectification.
Figure 3: Optimum layout for a gel to be quantified by SAFA.
Figure 4: Screenshot of the SAFA software and identification of the major software features.
Figure 5: Normalization scheme (Invariant Residue Normalization) applied to a Mg2+ titration of the P4-P6 subdomain of the T. thermophila group I intron.

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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.

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Authors and Affiliations

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

    Alain Laederach

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

    Alain Laederach & Russ B Altman

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

    Alain Laederach & Russ B Altman

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

    Rhiju Das

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

    Quentin Vicens

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

    Samuel M Pearlman

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

    Michael Brenowitz

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

    Daniel Herschlag

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  1. Alain Laederach
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Correspondence to Alain Laederach.

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Laederach, A., Das, R., Vicens, Q. et al. Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments. Nat Protoc 3, 1395–1401 (2008). https://doi.org/10.1038/nprot.2008.134

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