In certain experiments, it has been observed that RNA is often considered to be more fragile than DNA.The chemical instability of RNA and the ubiquitous presence of RNase make RNA extremely susceptible to degradation. Degraded RNA often leads to unsatisfactory results in downstream experiments. Therefore, after RNA extraction, it is necessary to perform quality tests on RNA to determine whether it meets the requirements of various downstream experiments, such as qPCR, Northern blot, cDNA library preparation, and cDNA labeling for microarray analysis.
Concentration (Purity) Detection
The yield of RNA is highly tissue-specific, determined by the abundance of RNA in different tissues and the difficulty of RNA extraction. Now, let’s take a look at the following two methods to measure the concentration of RNA.
01. Ultraviolet spectrophotometry
Nucleic acid molecules contain conjugated double bonds in their purine and pyrimidine rings that can absorb ultraviolet (UV) light and have a specific absorption peak at 260 nm. Most proteins contain aromatic amino acids, which have the maximum absorption at 280 nm. In addition, some salt ions and organic compounds have an absorption peak at 230 nm.
Figure 1. UV absorption spectra of different substances
The nucleic acid concentration can be measured using a UV spectrophotometer according to these physicochemical properties. In general, an OD260 of 1 is equivalent to 40 ng/μL of single-stranded RNA (ssRNA). Hence, the concentration of the RNA sample (ng/μL) is calculated as: OD260 × dilution factor × 40 ng/μL.
Note: The UV spectrophotometer should be calibrated with a solution that is used to dissolve RNA before measurement.
In addition to the concentration of RNA, OD260/280 and OD260/230 can indicate the purity of RNA:
Note: The solution pH can affect the measurement of OD value. For example, if ddH2O is used for elution, the solution is weakly acidic, which can increase OD280 and decrease OD260/280.
02. Fluorometric method (Qubit fluorometer)
Fluorescent dyes can specifically bind to target molecules (double-stranded DNA, single-stranded DNA, RNA, or protein) and emit fluorescence under the excitation of a light source at a specific wavelength. Then, the fluorometer reads the received fluorescence value and converts it into a concentration value for accurate quantification of DNA and RNA.
Figure 2. Qubit fluorometric quantification workflow
03. Comparison of two concentration measurement methods
Ease of use and fast workflow
Ease of use;
Precise distinction between DNA and RNA and more sensitive and accurate results;
Stable results free from interference of sample contamination, presence of salt ions, pH variation, and other related phenomena
Unable to distinguish between DNA, RNA, degraded nucleic acids, free nucleotides, and other impurities, resulting in falsely high concentration values; insufficient sensitivity
Higher cost; unable to directly distinguish degraded nucleic acids; unable to assay purity
UV spectrophotometry and the fluorometric method have specific advantages and are recommended to be used together. UV spectrophotometry can indicate the purity of nucleic acids. The fluorometric method delivers higher specificity and sensitivity, which is suitable for downstream experiments requiring precise quantification, such as next-generation sequencing, and for precious samples at low concentrations.
01. Agarose gel electrophoresis
The most common method used to assess the integrity of total RNA is to run RNA on an agarose gel. RNA electrophoresis can be run under either denaturing or non-denaturing conditions. When non-denaturing gels are used, it is difficult to determine the molecular weight accurately. Under denaturing conditions, ignoring the effect of the RNA secondary structure, the migration rate of RNA is linearly related to the logarithm of the molecular weight. Therefore, a denaturing gel is required to accurately determine the molecular weight of RNA molecules. However, an ordinary agarose gel is sufficient for a rapid assessment of RNA integrity.
Before reading the electropherogram, you need to understand the term sedimentation coefficient (S), which is a measure of the rate at which biomacromolecule sediments in a centrifugal field. A higher value indicates a larger molecule.
Figure 3. Example of electropherogram for high-quality RNA (liver)
What are the characteristics of a good electropherogram? (as shown in Figure 3)
(1) The electropherogram shows three rRNA bands from top to bottom. The top two bands are bright, clear, and sharp (i.e., the edges of the bands are clear):
★ For general animal samples, 28S, 18S, and 5S rRNA bands can be observed. If the extraction process involves column loading or CTAB+LiCl treatment, the 5S band may be darker or absent. For samples from insects or mollusks (e.g., Drosophila, oyster), there is only one distinct band.
★For general plant samples, three bands can be observed: 25S, 18S, and 5S;
★For general prokaryotes, three bands can be observed: 23S, 16S, and 5S;
Theoretically, 28S:18S is 2.7:1. However, a 2:1 ratio has long been used as a criterion for identifying intact RNA. In fact, almost all RNA extracted from most samples falls short of the 2:1 ratio. Using the DNA Marker as the control (Figure 3), if the 28S band at 2 kb and the 18S band at 0.9 kb are clear and 28S:18S > 1, the integrity of RNA can meet the requirements of most experiments.
(2) No redundant bands. There are two possibilities for miscellaneous bands: presence of DNA contamination (Figure 4) or multiple bands can be observed for the tissue type (e.g., samples containing a large number of chloroplasts, such as plant leaves).
Figure 4. DNA contamination
02. Agilent 2100 bioanalyzer assay
The Agilent 2100 bioanalyzer assay provides a precise concurrent digital assessment of RNA integrity, purity, and degradation via a lab-on-a-chip microfluidics platform, and can be used as an alternative to conventional gel assays. Its electrophoretic trace and RNA integrity number (RIN) are used to indicate RNA integrity.
Agilent 2100 bioanalyzer trace:
If the nucleic acid is intact, the baseline is flat. If the nucleic acid is severely degraded, the baseline is not flat, and more peaks indicating degradation appear.
The value reflects the RNA integrity. Within the range of 0 – 10, a larger value indicates greater RNA quality and integrity. The larger the RIN value, the flatter the baseline and the sharper the main peak. In general, RIN ≥ 5 indicates good RNA integrity, and RIN ≥ 8 indicates perfect RNA integrity.
The figure below shows a typical trace of a eukaryote generated by Agilent 2100 bioanalyzer.
Figure 5. Agilent 2100 bioanalyzer trace for eukaryote (HEK 293)
There are commonly five peaks in a typical electrophoretic trace of a eukaryote generated by Agilent 2100 bioanalyzer (Figure 5). However, not all eukaryotes have these peaks. For example, the organelles of plant leaves also contain ribosomes, and the three small peaks before 18S are for chloroplast RNA (Figure 6).
Figure 6. Bioanalyzer trace for rice leaf
(adapted from: Xie Yuelan et al. Chinese Agricultural Science Bulletin, 2020)
Details determine the quality of downstream experiment results! Mastering RNA assay methods will help you easily get started with experiments!
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