Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy

Sean R. Gallagher1

1 Analytik Jena, Upland, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3L
DOI:  10.1002/cpim.20
Online Posting Date:  February, 2017
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Abstract

Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. © 2017 by John Wiley & Sons, Inc.

Keywords: spectroscopy; DNA; RNA; quantitation; microvolume; absorbance; fluorescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Nucleic Acids Using Absorption Spectroscopy
  • Alternate Protocol 1: DNA Detection Using the DNA‐Binding Fluorochrome Hoechst 33258
  • Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence
  • Alternate Protocol 3: DNA Detection Using Picogreen dsDNA Quantitation Reagent
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Nucleic Acids Using Absorption Spectroscopy

  Materials
  • 1× TNE buffer (see recipe)
  • DNA sample to be quantitated
  • Calf thymus DNA standard solutions (see recipe)
  • Matched quartz semi‐micro spectrophotometer cuvettes (1‐cm pathlength)
  • Single‐ or dual‐beam spectrophotometer (ultraviolet to visible)

Alternate Protocol 1: DNA Detection Using the DNA‐Binding Fluorochrome Hoechst 33258

  • Hoechst 33258 assay solution (working solution; see recipe)
  • Calf thymus DNA standard solution (see recipe)
  • Dedicated filter fluorometer (Hoefer DQ 300, Promega QuantiFluor, or Invitrogen Qubit) or scanning fluorescence spectrophotometer (Shimadzu RF‐5301PC, Hitachi F‐2500, or Horiba FluoroMax, http://www.horiba.com)
  • Fluorometric square glass cuvettes or disposable acrylic cuvettes (Sarstedt)
  • Teflon stir rod appropriate for cuvettes
NOTE: The DNAQF Kit (Sigma‐Aldrich) contains the Hoechst 33258, buffers, and DNA standards for the assay.

Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence

  • Ethidium bromide assay solution (see recipe)

Alternate Protocol 3: DNA Detection Using Picogreen dsDNA Quantitation Reagent

  • PicoGreen dsDNA quantitation kit (Invitrogen) containing:
  • PicoGreen dsDNA quantitation reagent (Component A), 1 ml solution in DMSO (store frozen up to 6 months at –20°C, protected from light)
  • 20× TE (Component B), 25 ml of 200 mM Tris⋅Cl/20 mM EDTA, pH 7.5 (store up to 6 months at 4°C; may be frozen for long‐term storage)
  • Lambda DNA standard (Component C), 1 ml of 100 µg/ml in TE (store up to 6 months at 4°C; may be frozen for long‐term storage)
  • Spectrofluorometer or fluorescence microplate reader
NOTE: For either the kits or the stand‐alone reagent, sufficient reagent is supplied for 200 assays using an assay volume of 2 ml according to the protocol below. Note that the assay volume is dependent on the instrument used to measure fluorescence; with a microplate reader and a 96‐well microplate, the assay volume is reduced to 200 µl and 2000 assays are possible. The PicoGreen reagent supplied in the kits is exactly the same as the reagent sold separately. The DMSO stock solution should be stored frozen at –20°C and protected from light. The 20× assay buffer and lambda DNA standard in the kits are best stored at 4°C; however, either may be frozen for long‐term storage. When properly stored, components should be stable for at least 6 months.
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Figures

Videos

Literature Cited

   Applied Biosystems. 1987. User Bulletin Issue 11, Model No. 370. Applied Biosystems, Foster City, Calif.
  Cesarone, C.F., Bolognesi, C., and Santi, L. 1979. Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal. Biochem. 100:188‐197.
  Daxhelet, G.A., Coene, M.M., Hoet, P.P., and Cocito, C.G. 1989. Spectrofluorometry of dyes with DNAs of different base composition and conformation. Anal. Biochem. 179:401‐403.
  Desjardins, P.R. and Conklin, D.S. 2011. Microvolume quantitation of nucleic acids. Curr. Protoc. Mol. Biol. 93:3J:A.3J.1‐A.3J.16. doi: 10.1002/0470042727.mba03js93.
  Labarca, C. and Paigen, K. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344‐352.
  Le Pecq, J.‐B., 1971. Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. In Methods of Biochemical Analysis, Vol. 20 (D. Glick, ed.) pp. 41‐86. John Wiley & Sons, New York.
  Marmur, J. and Doty, P. 1962. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5:109‐118.
  Portugal, J. and Waring, M.J. 1988. Assignment of DNA binding sites for 4′,6‐diamidine‐2‐phenylindole and bisbenzimide (Hoechst 33258): A comparative footprinting study. Biochem. Biophys. Acta 949:158‐168.
  Stout, D.L. and Becker, F.F. 1982. Fluorometric quantitation of single‐stranded DNA: A method applicable to the technique of alkaline elution. Anal. Biochem. 127:302‐307.
  Van Lancker, M. and Gheyssens, L.C. 1986. A comparison of four frequently used assays for quantitative determination of DNA. Anal. Lett. 19:615‐623.
  Wallace, R.B. and Miyada, C.G. 1987. Oligonucleotide probes for the screening of recombinant DNA libraries. Methods Enzymol. 152:432‐442.
Key References
  Labarca and Paigen, 1980. See above.
  Contains a detailed description of the Hoechst 33258 fluorometric DNA assay.
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