Fluorometric Quantitation of RNA or DNA

  • 来源:生物谷
  • 时间: 2010/4/23
  • 浏览人数: 4748

    Materials
    DNA: Höchst 33258 (High range solution):  (Invitrogen H1398 or Sigma B1155) 1µg/mL in 1x TNE
        Filter and s
    tore at 4ºC in an amber bottle.
       
    10x TNE stock:  100 mM Tris, 2 M NaCl, 10 mM EDTA, pH7.4.  Store at room temp.
    RNA:  Ribogreen 200x (Invitrogen R-11491) detection reagent.  Store dessicated at -20ºC.
    Turner Designs TD-360 Fluorometer

    DNA: Long UV LED (underneath) and filter set (inside chamber)
    RNA: Blue LED and filter set
    RNA or DNA standards (100 ng/µL)
    TE pH 8 (DNA), pH 7.5 (RNA)

    Discussion
    Fluorometry utilizes fluorescent dyes which specifically bind DNA or RNA.  It requires a negative control (to set the zero point on the fluorometer) and a standard of known concentration.  The fluorometer shines light on the sample (excitation) and then measues level of fluorescent light being emitted to the side (at a 90º angle) of the excitation light beam.  The fluorescent dyes are relatively specific to nucleic acids as opposed to protein and other cellular components.  The fluorescence of the dyes increases when they bind nucleic acids.  Fluorometry is about 1,000x more sensitive than spectrophotometric absorbance (i.e. measurement of A260) and less susceptible to protein and RNA contamination.  However it also does not give a crude measurement of purity (like an A260/A280 ratio) nor does it assure that the DNA or RNA is not degraded (e.g. like size determination by gel electrophoesis).  Do not use glass (spectrophotmetry) cuvettes in a fluorometer because the frosted glass on the side of the cuvette interferes with detection of fluorescent light.

    Detection Ranges

    Large Cuvette
    Mini-cuvette
    High Range Solution
    20 ng - 1 µg
    1 - 50 ng
    Low Range Solution
    1 - 50 ng
    0.1 - 5 ng

    Procedure
    (High range assay solution)
    1. Insert the correct LED and filter set in the fluorometer for either RNA or DNA.
    2. Turn on the Fluorometer to warm up for 5 min.
    3. DNA:  Remove Höchst (high range) solution from the refrigerator.  Dilute Höchst 1/10 (v/v) in 1x TNE if a low range assay is being performed.
      RNA: Thaw Ribogreen and dilute to 1/200 (high range) or 1/2000 (low range) in 1x RNAse-free TE (pH 7.5).
      Make up enough reagent for the samples plus a blank and at least one control.
      • Large cuvettes require 1.5 mL/sample
      • Mini-cuvettes need 100 µL/sample.
      • Aliquot 1.5 mL (large cuvette) or 0.1 mL (microcuvette) of either Höchst or Ribogreen reagent into 1.5 mL eppendorf tubes. 
      • Add 1.5 µL of DNA or RNA to each tube and vortex.  In addition to samples this must also include a known standard and a TE or H2O control.
      • Using narrow (gel loading) tips pipet the TE or H2O control in a cuvette, close the chamber's lid and press the "Blank" button.  Press "1" when prompted to save the data.  Open the chamber's lid and transfer the control mix back to its original Eppendorf tube. 
      • Transfer the DNA or RNA standard to the cuvette and press the "Calibrate" button.  When prompted enter the (undiluted) concentration of the standard (i.e. 100) plus the "Enter" button. Press "1" when prompted to save the data.
      • Repeat the zero and calibration steps (6 and 7).  This is important to avoid negative values at low concentrations.
      • Measure each RNA sample and record the concentrations.
      Note:  The procedure for "Low range" assays is identical but it uses the detection reagent diluted 1/10x the cocentrations listed above to minimize background autofluorescence.