RNA Purification & Analysis

RNA purification is the process of preparing, isolating, extracting, and purifying RNA from a cell culture or tissue sample to obtain high-quality RNA for use in downstream applications. As biomolecules are first transcribed from DNA to RNA before becoming proteins, the functional complexity of various RNAs should not be understated.

Fig. 1

Simplified conformations of six common types of cellular RNA. Figure made in Biorender.

Primal RNA (priRNA) and messenger RNA (mRNA) are active members in transcription while transfer and transfer messenger RNAs (tRNA and tmRNA) are involved in translation. Additionally, small nuclear RNA (snRNA) function in splicing, while short interfering RNA (siRNA) and microRNA (miRNA) operate in post-transcription.

Purified RNA is commonly used as starting material for a number of experiments, including real-time and digital PCR, transcriptome analysis using next generation sequencing (NGS), and for the construction of cDNA libraries. Likewise, purified RNA may also be used in Northern blot, microarray analysis, or for use in nuclease protection assays. RNAs play vital and intricate roles in biology but in general, however, are fragile and may be difficult to recover. Adequate RNA purification is therefore essential to the success of downstream applications.

Common Extraction Methods

Spin Column Extraction

The spin column method is a solid phase extraction technique that relies on the fact that nucleic acids bind to solid silica under ideal conditions. The spin column method is an adsorption-based technique; it is based on the ability of RNA to create linkages to specific surfaces in the presence of chaotropic salts, rather, GTC/ GITC. This method offers quick test times, easy, simple steps, and commercial availability in many kit formats.

First, cells are lysed to free nucleic acids, then a binding solution, ethanol and the aqueous samples are added to the spin column.
A centrifugation step then forces binding of the solution through a silica gel membrane inside the spin column. As the solution is forced through the gel membrane, the freed nucleic acids will bind to the silica if the pH and concentration of the binding solution is optimal.
The nucleic acids will undergo a washing step, and then will undergo elution from the membrane where they can be collected from the bottom of the column.

Fig. 2

Prepacked ReadiUse™ Bio-Gel P6 Spin Column. The column was packed with P-6 DG in PBS buffer for sample volume 50~100 uL.

Phenol:Chloroform Extraction

The phenol:chloroform extraction method is a common technique used to purify RNA. Though this technique offers a simple, straightforward, cost-effective method of RNA purification, care should be taken upon each step that involves separating mediums, as upper and lower phases may become cross-contaminated.

Samples initially undergo a lysis step that utilizes a cationic detergent, commonly guanidinium thiocyanate or isothiocyanate (GTC or GITC) that effectively inactivates endogenous ribonucleases.
- Note: The addition of a low-pH phenol reagent, such as TRIzol or TRI, is used as a deproteinizing agent that further removes DNA from the sample.
Next, the sample undergoes an organic extraction step through the addition of chloroform. As chloroform is a purely organic reagent, it does not mix well with the cell lysate and the solution must be interspersed and then centrifuged, to separate the upper and lower phases, which correlate to the cell lysate and chloroform, respectively.
After, the cell lysate is isolated, isopropanol is added, and the sample will undergo another centrifugation step. In alcohol precipitation, the RNA will become pelleted towards the bottom of the test tube and the solution should be secondarily discarded. Multiple ethanol washes combined with additional centrifugation steps may be used to remove residual salts.
Finally, the purified RNA can be dissolved in RNase-free water or buffer, and stored for later or used immediately.

Other Methods

Other adsorption methods aside from spin column extraction exist as well, including those that utilize:

magnetic beads
polystyrene latex materials
cellulose matrices
glass fibers

In principle, the procedures are similar; the samples are lysed, exposed to an adsorbing material, mixed to facilitate binding, washed to remove contaminants, then sedimented.

Fig. 3

Illustration of the principles of isopycnic centrifugation, showing the transition of the starting mixed sample to the equilibrium state of sample particles settled into layers of equivalent density. Even if centrifugal spinning continues after the equilibrium point, the particles will not move out of these layers into a pellet, minimizing the risk and sample damage of 'overspinning'. Figure made in BioRender.

These techniques also come with some drawbacks. Loss of the membrane or beads as well as overdrying must be carefully avoided, as these occurrences will result in a loss of RNA. Caution should also be taken when separating phases after centrifugation steps, and separation techniques should involve the use of a long flexible pipette over an aspirator.

Other techniques of RNA purification include the isopycnic gradient method, which is a density driven technique that similarly utilizes GTC/ GITC, a column, and centrifugation. This method may commonly be used for isolating RNA free of proteins, DNA, polysaccharides and other cellular components. It may be desired instead that mRNA be purified from the total RNA sample. To do so, the chemical structure of mRNA using the polyadenylate tail located at the 3' terminus is exploited, and mRNA may be obtained by chromatographic methods, or by the use of a magnetic field.

Additionally, various kits are commercially available for the extended purification of other RNA species from total RNA, including those for small RNAs (like miRNA and siRNAs), cell-free mRNAs, or even for the simultaneous co-purification of RNA and proteins together.

Common Analysis Methods

Ultraviolet (UV) Spectroscopy

Common analysis techniques involve the use of UV spectroscopy, where diluted RNA can be measured between 260-280 nm, representing a 260/280 nm ratio of absorbance. Nucleic acid concentration is calculated using the Beer-Lambert law, which predicts a linear relationship between absorbance and concentration.

UV spectroscopy does not discriminate between RNA and DNA, so a sample treatment step with RNase-free DNase to remove contaminating DNA prior to analysis is vital. It is important to note that other contaminants, including residual proteins and/or phenol, can interfere with absorbance readings, so optimal extraction and purification steps may be necessary.

The 260/280 nm absorbance ratio is dependent on both pH and ionic strength; as the pH of the sample increases, the 280 nm absorption decreases and the absorption at 260 nm is unaffected, leading to an increased ratio. Since water is often acidic, it may lower the 260/280 nm ratio, so a buffer with a slightly alkaline pH, like Tris-EDTA, is recommended for use as a diluent and blank to provide reproducible readings.

Sample readings are also taken using quartz cuvettes, so care must be taken to ensure these apparatuses are cleaned thoroughly as dirt and dust may impact absorbance at 260 nm. Background corrections may also need to be performed using readings from a blank at 320, 260, and 280 nm.

Related Resources

Fluorescent Dyes

Fluorescent dyes may also be used for analyzing RNA purification, which utilize the excitation, or binding, of fluorophores to RNA to evaluate the purity of the RNA test sample. Sample fluorescence can then be plotted against a standard curve formed from known concentrations at 260 nm. Detection and quantitation can be performed using a laboratory standard or filter fluorometer, or a fluorescence microplate reader.

Fig. 4

live and fixed HeLa cells stained with StrandBrite RNA Green and Hoechst 33342 or DAPI

Fluorescence images of live and fixed HeLa cells stained with StrandBrite RNA Green (Green) and counter-stained with Hoechst 33342 or DAPI (Blue). Fluorescence signal were measured using a fluorescence microscope with FITC filter.

Note: To ensure the accuracy of fluorometric readings, possible contaminants must be carefully assessed and removed where possible. For fluorometric methods, continuous freeze-thawing of the sample and experimental reagents must also be avoided.

Agarose/Acrylamide Gel Electrophoresis

In agarose and acrylamide gel electrophoresis samples are loaded into precast gels then stained with fluorescent dyes that bind nucleic acids. After the addition of an electrical current, nucleic acid fragments move through the gel and are separated on the basis of size. Larger fragments move more slowly while smaller fragments move more quickly through the gel, and separated fragments may be visualized by exciting the fluorescent dye bound to the nucleic acid.

Qualitative RNA concentrations may be measured by comparing the fluorescent intensity of the sample RNA bands to the known standards, run alongside the test samples. Quantitative assessment may be performed by equipment with built-in software to analyze an image of the gel, a technique called gel densitometry.

Tools

RNA Concentration Calculator
Use our RNA Concentration Calculator to determine the concentration of RNA in a solution.

Real-Time Quantitative PCR (qPCR) and Reverse Transcriptase PCR (RT-qPCR)

In qPCR the amount of amplified product is measured at the end of each cycle after amplification, or in real-time during the exponential phase of amplification. The incorporation of a reverse transcriptase step to qPCR allows the qualitative measurement of the amount of each specific RNA species in a sample.

Fig. 5

One-step RT-PCR diagram. In one-step RT-PCR, cDNA synthesis via reverse transcription (RT) and subsequent PCR amplification occur in the same reaction vessel (figure made in BioRender).

Here, amplified products are also measured through use of fluorescent probes. Adequate primer design is crucial to the success of the experiment, and RNA-specific primers that flank an intron of the target sequence should be used for cDNA detection. Primers within an intron can also be used to detect DNA contamination in an RNA sample. The use of multiple fluorophores, separately labeled to different primers may also be preferred. This technique allows researchers to analyze multiple targets in a single reaction and examine samples for the presence of PCR inhibitors.

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