Protocol for RNA Quantitation using Ethidium Bromide (etbr)

Robert W. Fisher, Department of Radiation Oncology and the Curriculum in Toxicology, University of North Carolina at Chapel Hill, USA.

1. Introduction

Detection and quantitation of nucleic acids is an essential technique in the molecular biology laboratory. Nucleic acids are commonly stained with fluorescent dyes when separated on the basis of size in agarose gels. These fluorescent dyes are also used to quantitate nucleic acids in solution. The high sensitivity and selectivity of fluorometric assays over spectrophotometric methods has led to the development of instruments designed to quantitate nucleic acids based on their interactions with fluorescent dyes. There are a multitude of fluorescent nucleic acid dyes such as ethidium bromide, propidium iodide, SYBR™ Green I and II, PicoGreen™ , and Hoechst 33258 Dye. Some of the newer dyes offer sensitivities in the picogram range. These fluorochromes allow the quantitation of DNA in the presence of contaminating protein. The most sensitive dyes are often specific for double-stranded nucleic acids over single-stranded nucleic acids. This specificity is useful if there is a high level of single-stranded RNA present to interfere with the measurement. On the other hand, the need to quantitate RNA often arises in the molecular biology laboratory and the selectivity of the DNA specific dyes becomes a severe handicap.

One way around this problem is to use a dye that binds to nucleic acids in general, whether they be single- or double-stranded. Probably the most common dye used for this purpose is ethidium bromide. Ethidium bromide is a polycyclic fluorescent dye that binds to double-stranded DNA molecules by intercalating a planar group between the stacked base pairs of the nucleic acid. Ethidium bromide can also bind to secondary structure in single-stranded RNA molecules: regions of local base pairing offer the stacked base pairs necessary for the dye molecules to intercalate. When excited by light at or near 546 nm, the dye-nucleic acid complex exhibits an increased (about 20 fold) fluorescent yield at an emission wavelength of 590 nm.

The following describes an ethidium bromide based fluorometric method for quantitation of RNA solutions using a Turner BioSystems TD-700 Laboratory Fluorometer.

2. Materials Required

• TD-700 Fluorometer with standard PMT and 10 mm x 10 mm square cuvette adaptor (P/N 7000-009)
• Clear Quartz Lamp (P/N 10-046) or Quartz-halogen lamp (P/N 7000-930)
• Excitation Filter 550 nm (P/N 034-0500)
• Emission Filter >570 nm (P/N 10-052R)
• 10 mm x 10 mm Glass Square Cuvettes (P/N 7000-955)
• Transfer RNA (Sigma Chemical)
• DEPC H2O or Ultrapure H2O
• 10X TNE Buffer Stock Solution (100 mM Tris, 2 M NaCl, 10 mM EDTA, pH 7.4)
• Ethidium Bromide (5-10 mg/mL)

3. Experiment Protocol

Three series of experiments were designed to:

1. Test the linearity and reproducibility of RNA measurements under the conditions used for DNA quantitation using Hoechst 33258 Dye; 2. Optimize the ethidium bromide concentration used for RNA quantitation; and 3. Determine whether a typical laboratory sample of total RNA can be quantitated.

Fresh 1X TNE buffer was diluted from the stock 10X TNE buffer daily. Immediately before use, assay buffer was prepared by adding ethidium bromide to the 1X TNE buffer to achieve a final concentration of 0.1 ug/mL, 1.0 ug/mL, or 10 ug/mL. Assay buffer was kept in a light-proof container during use. In each experiment, 2 mL of the assay buffer was used to zero the fluorometer. Two uL of each tRNA sample was added to 2 mL of the assay buffer, mixed, and inserted into the fluorometer for measurement.

In the first three experiments, standard solutions of transfer RNA (tRNA) were prepared by dissolving tRNA in DEPC-treated H2O to a final concentration of 3.724, 1.790, or 1.742 mg/mL as verified by measuring the A260 on a Beckman DU70 Spectrophotometer. In the first experiment, three separate sets of serial dilutions were prepared to determine reproducibility. In the second and third experiments, each one of a set of serial dilutions was measured in triplicate.

In the final experiment, total RNA was isolated from human mammary epithelial cell (HMEC) line 184B5 using the acid guanidinium thiocyanate procedure of Chomczynski and Sacci. The precipitated total RNA was dissolved in DEPC-treated H2O to a final concentration of 1.700 mg/mL as verified on a spectrophotometer. Fluorometric measurements were made using an ethidium bromide concentration of 1.0 µg/mL.

All measurements were made at room temperature on a Turner BioSystems TD-700 Fluorometer. The instrument was operated in the multi-optional mode, measuring raw fluorescence. The sensitivity was auto-set as described in the operating manual. Standard precautions were taken to avoid RNase contamination problems.

4. Results

1. Determining Reproducibility & Linearity: The first experiment was designed to determine the reproducibility of RNA measurements and to verify whether a linear concentration vs. fluorescence response occurs under conditions similar to those used in measuring DNA samples with Hoechst 33258 Dye. The 3.724 mg/mL standard tRNA solution was used to prepare three separate serial dilutions with final concentrations of 2.000 mg/mL, 1.000 mg/mL, and 0.500 mg/mL. The final ethidium bromide concentration in the assay buffer was 1.0 µg/mL. Each serial dilution was measured once and a linear concentration vs. fluorescence curve was obtained, yielding an r2 value of 0.983 (Figure 1).

   In order to test linearity at lower RNA concentrations, tRNA samples from 1.790 µg/mL down to 112 ng/mL were measured. Each sample was measured in triplicate, and again the results were highly linear (r2 = 0.999) (Figure 2).

   
   Figure 1. Transfer RNA Concentration vs. Fluorescence Response

   
   Figure 2. Fluorescence Response of Transfer RNA down to 112 ng/mL

2. Optimizing Ethidium Bromide Concentration: The next experiment was designed to determine the optimal concentration of ethidium bromide. One µg/mL ethidium bromide was chosen in the preceding experiments because it is at the high limit of concentrations used to stain agarose gels for RNA. Final tRNA concentrations of 1.742 µg/mL, 0.871 µg/mL, 0.436 µg/mL, 0.218 µg/mL and 0.109 µg/mL were measured in triplicate using 10 µg/mL, 1.0 µg/mL and 0.1 µg/mL of ethidium bromide in the TNE buffer. The results, as shown in Table 1 and Figure 3, indicate an optimal ethidium bromide concentration of 1.0 µg/ml. Using 1.0 µg/ml of ethidium bromide one achieves high reproducibility, strong linear response, and sufficiently low background fluorescence to measure less than 100 ng/mL RNA.

   
   Table 1. Fluorescence of Transfer RNA Using Varying Concentrations of Ethidium Bromide

   
   Figure 3. Fluorescence of Transfer RNA using different EtBr concentrations.

3. Measuring Total RNA: Finally, it was determined that a typical laboratory sample of total RNA can be quantitated using the TD-700 Fluorometer. Total RNA from human mammary epithelial cells was isolated. Total RNA concentrations of 1.700µg/ml, 0.850µg/ml, 0.425µg/ml and 0.213µg/ml were measured. Figure 4 shows the fluorescence response of these samples.

   
   Figure 4. Fluorescence of HMEC total RNA using 1.0µg/mL EtBr.

5. Discussion

The experimental results demonstrate that the Turner BioSystems TD-700 Fluorometer can quantitate both transfer and total RNA using ethidium bromide as the fluorochrome. The procedure is fast, simple, and can be done using a minimum sample volume. An additional benefit is that the same TNE buffer can be used to measure DNA concentrations, changing only the filter sets and substituting Hoechst 33258 Dye for ethidium bromide in the assay buffer (See Turner BioSystems Application Note "A Method for DNA with Hoechst 33258 Dye and Thiazole Orange").

It should be noted, however, that a standard curve be generated using RNA similar to the RNA to be analyzed. For example, an accurate measurement of total RNA would not be possible using a standard curve generated from transfer RNA. On the other hand, if a number of samples are to be compared to each other (to normalize gel loading, for example) then a transfer RNA standard should be adequate.

6. References

1. F.A. Ausubel, et al., Eds., Current Protocols in Molecular Biology (John Wiley & Sons, New York, © 1996.
2. P. Chomczynski, N. Sacchi, Anal.Biochem. 162, 156-159, © 1987.
3. J.B. LePecq, C. Paoletti, Anal. Biochem. 17, 100-107, © 1966.
4. P.N. Gray, G.F. Saunders, Biochim. Biophys. Acta 254, 60-77, © 1971.
5. M.G. Murray, H.E. Paaren, Anal.Biochem. 154, 638-642, © 1986.
6. K. VanDyke, C. Szustkiewicz, Anal.Biochem. 23, 109-115, © 1968.

Note: To optimize RNA quantitation, Turner BioSystems tested various lamp and filter configurations. Using the following, we were able to maintain linearity down to 50 ng/mL tRNA:

• Daylight White Lamp (10-045) with excitation filter 034-0500 and emission filter 10-052
• 10 x 10 mm methacrylate cuvettes (7000-959)
• 3 mL sample volume