Here we discuss three common methods used to quantitate RNA and tips for optimizing each of these methods.
The Agilent 2100 bioanalyzer uses a combination of microfluidics, capillary electrophoresis, and fluorescent dye that binds to nucleic acid to evaluate both RNA concentration and integrity. An RNA reference standard (the RNA 6000 Ladder Cat# 7152; Ambion) and a microfluidics chip (The RNA Lab Chip; Agilent Technologies) are also required. The RNA 6000 Ladder is composed of six RNAs ranging in size from 0.2 6 kb. The ladder and samples are loaded in designated wells on the RNA Lab Chip. Size and mass information is provided by the fluorescence of RNA molecules as they move through the channels of the chip. The instrument software automatically compares the peak areas from unknown RNA samples to the combined area of the six RNA 6000 Ladder RNA peaks to determine the concentration of the unknown samples. The RNA 6000 Nano System has a broad dynamic range and can quantitate between 25 500 ng/ml of RNA with a covariance of ~10%.
Perhaps the most powerful feature of the Agilent 2100 bioanalyzer is its ability to provide information about RNA integrity. As each RNA sample is analyzed, the software generates both a gel-like image and an electropherogram (Figure 3). When analyzing total RNA, the areas under the 18S and 28S ribosomal RNA peaks are used to calculate the ratio of these two major ribosomal RNA species and these data are displayed along with quantitation data on individual electropherograms (Figure 3a). Significant changes in the ratios of the 18S and 28S ribosomal RNA peaks are indicative of degraded RNA.
Figure 3. Agilent 2100 Bioanalyzer Electropherograms of RNA Samples.
A. Electropherogram of a Total RNA Sample.
Total RNA (100 ng) was analyzed on an Agilent 2100 bioanalyzer. The resulting electropherogram shows the characteristic signature of a high quality total RNA sample.
B. Electropherogram of Amplified aRNA Sample.
Total RNA 2 µg corresponding to 60 ng mRNA) was amplified using the MessageAmp aRNA Kit (Ambion Cat# 1750) resulting in (90 µg aRNA, a 1500 fold amplification. The aRNA (900 ng) was analyzed on an Agilent 2100 bioanalyzer. The resulting electropherogram shows the classic output of a high quality aRNA sample.
In addition to its usefulness for analysis of total RNA, the bioanalyzer is also a superior tool for analyzing mRNA and amplified aRNA (antisense RNA) integrity. Intact mRNA and aRNA profiles consist of a broad distribution of signal, with the bulk of the RNA usually falling between 1 and 2 kb, though this will vary from tissue to tissue (Figure 3b). A significant shift of the profile towards lower molecular weights is indicative of poor RNA integrity.
- The area of the peaks derived from the RNA 6000 Ladder is used as a mass standard for unknowns, so accurate quantitation of your unknowns is dependent on careful handling of this standard. We recommend that the RNA 6000 Ladder be thoroughly mixed and carefully pipetted to reduce error. For best performance, the standard should be aliquoted into non-stick, nuclease-free tubes to avoid multiple freeze-thaw cycles of a single stock tube.
- Quantitation is affected by ionic strength of the sample, which can quench fluorescence in RNA samples. Therefore, when possible, RNA should be suspended in nuclease-free water to minimize differences between the RNA 6000 Ladder and the sample to be measured. If this is not possible, be aware that the unknown concentration may be underestimated.
- Generally, we find that some 23S and 28S rRNAs do not migrate according to their molecular weights. For example, mammalian 28S rRNA, 4.8 kb in length, consistently migrates just ahead of the 4 kb peak in the RNA 6000 Ladder. This is likely due to the highly structured nature of 23S and 28S rRNAs.
- Although this assay has a broad linear range (~25 ng 500 ng) overloading the chip with RNA can affect performance of the RNA Lab Chip. For consistent results, we recommend loading 50 ng 250 ng of RNA.
- The fluorescent dye is light sensitive, so store dye concentrate and working solutions away from light; e.g. wrap tubes in foil.
- Follow the manufacturer's recommendations for maintenance of the electrodes and the priming station. Poor Lab Chip Loading (priming) and formation of salt bridges between electrodes are common causes of poor assay performance.
Wilfinger WW, Mackey K, and Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22:474 481.