Isolating high-quality RNA is the most critical step for successfully performing a broad range of assays, from RT-qPCR or microarray analysis to cDNA library preparation, as well as Northern blot studies. It is even critical for high-throughput transcriptome analysis using next-generation sequencing techniques.
Therefore, getting the most from your RNA isolation procedure is a must. High-quality experiments require high-quality samples, and maximizing yield of non-degraded RNA isolation is key. In this article, we will discuss three of the most common RNA extraction techniques and go over the pros and cons for each strategy.
The organic extraction method
Organic extraction of nucleic acids is historically the most common, tried-and-true method for RNA isolation and removing cellular proteins. This technique requires homogenization of your sample in a phenol-containing solution (usually phenol-chloroform). The phenol-chloroform mixture is immiscible with water, therefore when centrifuged, the samples form two distinct phases.
The lower (organic) phase and phase interface contain denatured proteins, while the less-dense upper (aqueous) phase contains nucleic acids. Importantly, the phase extraction of DNA and RNA is pH-dependent, when the pH is greater than 7.0, both RNA and DNA will resolve in the aqueous phase. A pH less than 7.0, DNA more readily denatures and precipitates into the organic phase and phase interface, with RNA remaining in the aqueous phase.
The aqueous phase containing your RNA is then carefully removed by pipetting (with care not to touch the interface or organic phase, as this can contaminate your sample) and RNA is then precipitated with alcohol and rehydrated for further analysis.
The pros:
- Organic extraction is the gold standard.
- Protocols are well-established and routinely used, making the procedure straightforward for novice researchers.
- Proteins are rapidly denatured and RNA is quickly stabilized.
- The process is applicable to larger samples (such as human or animal tissues) as well as smaller samples from cell culture based experiments.
The cons:
- Not very amenable to high-throughput processing and difficult to automate.
- Manual processing of samples can be laborious.
- Use of hazardous chemical and chlorinated organic waste must be managed carefully.
The spin column extraction method
This is a solid phase extraction technique to bind and isolate RNA within filter-based spin columns. These spin columns utilize membranes that contain silica or glass fiber to bind nucleic acids. Samples are lysed in a buffered solution containing RNase inhibitors and a high concentration of chaotropic salt. The lysates are passed through the silica membrane using centrifugal force, with the RNA binding to the silica gel at the appropriate pH.
The membrane containing residual proteins and salt is then washed to remove impurities, with flow-through discarded. RNA is subsequently eluted with RNase-free water, as RNA is stable at a slightly acidic environment.
The pros:
- Simple, straightforward procedure to perform.
- A ready to use kit format, which adds convenience.
- Amenable to large-scale and high-throughput processing, including automated methods.
- Flexible for use with both centrifugation or vacuum based systems.
The cons:
- Starting with too much sample or incomplete homogenization can clog the membrane and/or result in contamination with proteins or genomic DNA.
- Incomplete cellular lysis can lead to low yields.
- Automation systems for centrifugation or vacuum can be expensive and complex to set up.
Magnetic particle extraction method
This strategy for bioseparation utilizes beads with a paramagnetic core (in other words, they have properties of magnetism only when in proximity to an external magnetic field) coated with, most commonly, a matrix of silica for binding nucleic acids. In this method, cells are lysed in a buffer with RNase inhibitors and then incubated with the magnetic beads, allowing the particles to bind RNA molecules.
The magnetic beads can then be quickly collected by being placed in proximity to an external magnetic field. The supernatant is removed and then subsequently washed and resuspended with removal of the magnetic field. This process can be easily repeated for multiple washes. The RNA is eluted from the magnetic beads with RNase-free water into solution, and the supernatant (containing the pure RNA) can then be transferred.
The pros:
- RNA isolation technique is most amenable to automation and high-throughput methods.
- The magnetic collection and resuspension steps are rapid and simple to perform.
- Rapid and simple magnetic collection and resuspension steps.
- Non-filter method reduces concern for clogging.
- No organic solvent hazardous waste.
The cons:
- Viscous samples can impede migration of magnetic beads.
- While more easily amenable to automation, this technique can be laborious when performed manually with large numbers of samples.
- Risk of contamination of RNA samples with residual magnetic beads.