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  • Evaluation of Reaction Products (E2040)

    Protocol

    1. Quantification by UV Light Absorbance

      RNA concentration can be easily determined by measuring the ultraviolet light absorbance at 260 nm wavelength, however, any unincorporated nucleotides and template DNA in the mixture will effect the reading. Free nucleotides from the transcription reaction must be removed before the RNA concentration can be quantified. A 1:200 dilution of a sample of the purified RNA should give an absorbance reading in the linear range of a spectrophotometer. RNA dilution may not be necessary if using a NanoDrop™ Spectrophotometer. A NanoDrop™ Spectrophotometer can read RNA concentrations from 10 ng/μl to 3000 ng/μl directly. For single-stranded RNA, 1 A260 is equivalent to RNA concentration of 40 μg/ml. The RNA concentration can be calculated as follows:

      A260 x dilution factor x 40 = __ μg/ml RNA


    2. Analysis of Transcription Products by Gel Electrophoresis

      To evaluate transcript length, integrity and quantity, an aliquot of the transcription reaction should be run on an appropriate denaturing agarose gel or polyacrylamide gel. Transcripts larger than 0.3 kb can be run on agarose gels, whereas denaturing polyacrylamide gels (5–15%) are necessary for smaller transcripts. The gels should be run under denaturing conditions to minimize formation of secondary structure from the transcript.
      1. Preparation of denaturing gels
        1. Denaturing agarose gel:
          To make 100 ml 1% denaturing agarose gel, add 1 gram agarose powder to 72 ml nuclease-free water. Melt the agarose, add 10 ml 10X MOPS buffer. Then in a fume hood, add 18 ml fresh formaldehyde (37%), mix well. Pour the gel. 10X MOPS gel running buffer: 0.4 M MOPS (pH 7.0), 0.1 M Sodium Acetate, 10 mM EDTA
        2. Denaturing PAGE/Urea Gel:
          5–15% PAGE/Urea gel. We recommend using commercially available premade gels. Use standard TBE gel running buffer. 10X TBE buffer: 0.9 M Tris Base, 0.9 M Boric Acid, 20 mM EDTA 
      2. Gel electrophoresis of non-radiolabeled RNA
        1. Mix 0.2–1 μg RNA sample with an equal volume of RNA Loading Dye (2X) (NEB #B0363 ).
        2. Denature the RNA sample and an aliquot of RNA marker by heating at 65–70°C for 5–10 minutes.
        3. Pulse-spin prior to loading onto gel. d. Visualizing RNA by staining the gel with SYBR Gold or ethidium bromide.
      3. Gel electrophoresis of radiolabeled RNA
        1. Mix an aliquot of labeled RNA with an equal volume of RNA Loading Dye (2X) (NEB #B0363).
        2. Denature the RNA sample by heating at 65–70°C for 5–10 minutes.
        3. Pulse-spin prior to loading onto gel.
        4. Visualizing RNA by autoradiography.
      Agarose gels should be dried before exposing to X-ray film, but thin (< 1 mM thickness) polyacrylamide gels can be transferred to filter paper, covered with plastic wrap and exposed to X-ray film directly (when 32P is used). Exposure time could range from 20 minutes to overnight depending on the specific activity of the RNA probe and the type of intensifying screens used. Exposure time could be much shorter if the gels are exposed to Storage Phosphor Screen (GE or equivalent).