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Matthew W. Fields, Section of Microbiology, Cornell University, Ithaca, New York 14853
Introduction
Biologists frequently monitor the effect of environmental stimuli on living
organisms by quantitating mRNA levels. The relative abundance of mRNA
is typically determined with probes and hybridization blots, but these
methods depend on accurate and sensitive methods for measuring total RNA.
When fluorochromes bind to nucleic acids, fluorescence occurs, and changes
can be monitored with a fluorometer. The following section describes the
quantitation of total RNA with a VersaFluor fluorometer (Bio-Rad).
Materials and Methods
Total RNA was isolated from the strictly anaerobic ruminal bacterium,
Prevotella bryantii, using an acid-phenol extraction procedure.1 All solutions,
tubes, and pipets were DEPC treated and autoclaved before use. Escherichia
coli K12 total RNA was isolated using a commercially available RNA purification
kit and was stored at -70 C. RNA concentrations were determined using
the VersaFluor fluorometer and RiboGreenTM RNA quantification kit (Molecular
Probes, Inc). The excitation was at 490 nm (EX 490/10 filter), and the
emission was monitored at 520 nm (EM 520/10 filter). A standard curve
was generated with E. coli ribosomal RNA (supplied with the kit), and
the range was 1 to 50 ng/ml.2 The standard and all samples were diluted
in TE buffer and mixed with RiboGreen (Molecular Probes, Inc.) that had
been diluted 2,000-fold. Fluorescence emission intensity was measured
using disposable cuvettes, and the gain and range settings were adjusted
to medium and 10,000, respectively. Relative fluorescence units (RFU)
were recorded and plotted as a function of RNA added (Figure 1, R2=.988),
and this linear relationship was used to determine the RNA concentration
of samples.
When E. coli ribosomal RNA was treated with DNase-free RNase3 for 1 hour at 37 C, no RFU was detected. If samples were treated with DNase-free RNase, there was a 97% decrease in RFU. These results corroborated the idea that fluorescence emissions were being generated by RNA and not DNA.
RNA quantitation is a crucial feature of many molecular measurements. Prevotella bryantii endoglucanase mRNA was detected with slot-blots using a fluorochrome-labeled probe (Figure 2). Accurate quantitation of RNA was critical, and the VersaFluor fluorometer was used reliably in determining RNA concentrations.
References
1. Wen, Z. and Morrison, M., AEM, 62, 38263833 (1996).
2. Molecular Probes, Inc. Product Information Sheet, RiboGreen RNA quantitation reagent (1997).
3. Ausubel, F., et al., Short Protocols in Molecular Biology. A1-51 (1997).
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