Quantification of RNA

Quantification of the final product is an essential step for many applications to ensure similar RNA loading across subsequent assays. Four methods are commonly used for assessing the quantity and quality of the RNA extracted:

1. UV spectroscopy.

2. Fluorescent dyes (RiboGreen, Molecular Probes).

3. Denaturing gel electrophoresis.

4. Microfluidic approaches.

3.5.1. UV Spectroscopy

Nucleic acids strongly absorb light in the UV region of the spectrum, and UV spectroscopy has, as such, become the traditional approach to assessing the concentration and purity of RNA. The concentration of nucleic acid is calculated using the Beer-Lambert law, predicting a linear change in absorbance with concentration. An A260 reading of 1 is equivalent to 40 ^g/mL of single-stranded RNA.

RNA purity may be assessed by the A260/A280 ratio, with high-purity RNA possessing a ratio between 1.8 and 2.0. It should be noted that the A260/A280 ratio is also dependent on both pH and ionic strength. As the A280 decreases with pH (whereas the A260 is unaffected), this may lead to a lower A260/A280 ratio when using acidic diluent. Using a buffered solution with a slightly alkaline pH (such as TE, pH 8.0) will therefore provide more accurate and reproducible readings (11).

Spectroscopy has several failings, which many users do not stop to consider. First, this technique offers no measure of RNA quality. Many users confuse purity with quality, but spectroscopy offers no indication of whether the RNA is intact or degraded. Second, spectroscopy is accurate over only a limited dynamic range of concentrations (1).

3.5.2. Fluorescent Dyes

Several fluorescent dyes are available that demonstrate a large increase in fluorescence when bound to nucleic acids. By use of a fluorometer, these dyes enable comparisons to be made against known concentrations of RNA. Information regarding these dyes and the necessary protocols are available from suppliers (see Note 8). Although they are insensitive to non-nucleic acid contaminants, again these approaches offer little in the way of quality assurance.

3.5.3. Gel Electrophoresis

The integrity of total RNA may be confirmed by gel electrophoresis, enabling the 18S and 28S rRNA species to be visualized. Although a nondenaturing gel may be used, most forms of RNA possess extensive secondary structure that prevents them from migrating strictly according to size (in a similar way that supercoiling may affect plasmid migration size). Precise quantification of RNA is difficult with electrophoresis, although the ability to detect the integrity of rRNA offers a distinct advantage over spectroscopy.

A problem with denaturing gel electrophoresis is that it necessitates the use of formaldehyde, which is toxic by inhalation and is also classified as a carcinogen. Use of denaturing gels therefore requires a separate electrophoresis setup to limit formaldehyde exposure, as well as to prevent RNAse contamination. For these reasons, coupled with increased availability of lab-on-a-chip systems, denaturing gel electrophoresis has become a less frequent approach to RNA analysis.

For simple detection of 18S and 28S bands (at around 1.9 and 5 kb in size, respectively), nondenaturing agarose gel electrophoresis may suffice. In this case a 1% agarose gel loaded with around 1 ^g of total RNA should enable detection of the two larger rRNA bands, indicative of intact RNA.

The availability of lab-on-a-chip systems, exemplified by the Agilent 2100 bioanalyzer, enables a new approach to RNA quantification, and most important, a single method of enabling concentration and RNA integrity to be measured simultaneously (see Note 9). The bioanalyzer utilizes a combination of microfluidics, capillary electrophoresis, and fluorescent dyes, recording the fluorescence of RNA as it migrates through the channels of the chip. The microfluidic approach offers a major advantage in that it requires much smaller volumes of RNA. The output is usually represented as an electropherogram, within which the 18S and 28S ribosomal RNA species are clearly visible as large peaks (Fig. 4A). By calculating the area under the curve of these major rRNA species, the integrity of the RNA may be calculated, with degraded RNA producing a shift toward lower molecular weights (Fig. 4B).

4. Notes

1. Additional information on solid phase kits may be found on the following supplier websites: Ambion (www.ambion.com); Amersham (www5.amersham biosciences.com); Invitrogen (www.invitrogen.com); Qiagen ( www.qiagen. com); and Sigma-Aldrich (www.sigmaaldrich.com). In addition, the Ambion website offers an excellent library of RNA related resources: www.ambion.com/ techlib/basics/rnaisol/index.html.

2. DEPC is a highly reactive alkylating agent, used to elimate RNases from solutions, glassware, and plasticware. DEPC is a potent protein denaturant and a suspected carcinogen. As such, wear appropriate gloves and lab coat and use only in a fume hood. Although reverse-osmosis systems are typically free of RNases, microbial growth may result in contamination, particularly in centralized sys-

Fig. 4. Analysis of RNA quality using the Agilent Bioanalyzer 2100, in which 1 ^L of total RNA was run, enabling the 18S and 28 bands to be visualized as an electro-pherogram. The 28S/18S ratio should be around 2 for high-quality RNA, with a flat baseline (A). RNA degradation is visible as a decrease in the two ribosomal RNA peaks with a corresponding increase in smaller RNA degradation products, resulting in a noisier baseline (B).

Fig. 4. Analysis of RNA quality using the Agilent Bioanalyzer 2100, in which 1 ^L of total RNA was run, enabling the 18S and 28 bands to be visualized as an electro-pherogram. The 28S/18S ratio should be around 2 for high-quality RNA, with a flat baseline (A). RNA degradation is visible as a decrease in the two ribosomal RNA peaks with a corresponding increase in smaller RNA degradation products, resulting in a noisier baseline (B).

tems. To treat H2O, add 0.1% DEPC at 37°C for 1 h, or overnight at room temperature. The DEPC is then inactivated by autoclaving on a liquid cycle (1).

3. RNAZap® is available from Ambion, RNAZap™ Sigma-Aldrich, and most molecular biology suppliers.

4. For details, see the Q-biogene (www.qbiogene.com/fastprep/index.shtml) or Qiagen websites.

5. Important safety note: Guanidium salts are chaotropic agents that destroy the three-dimensional structure of proteins (hence inhibiting RNases) and are therefore dangerous. Phenol is toxic and extremely corrosive. Handle with care, and wear gloves, lab coat, and eye protection at all times.

6. With small tissue samples (<5 mm), 0.5 mL of lysis reagent works well. For larger samples, increase the volume of chloroform and isopropanol proportionally. For RNA-rich tissues such as liver, 1 mL of lysis reagent is recommended.

7. RNAsecure™ (Ambion) perfoms well for resuspension of RNA pellets. For further information, see the Ambion website: www.ambion.com/catalog/ CatNum.php?7005.

8. See, for example, RiboGreen from Molecular Probes (www.probes.com/).

9. For details on the RNA LabChip® and 2100 Bioanalyzer, see the Agilent website: www.chem.agilent.com/Scripts/PCol.asp?lPage=50.

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