The Basics of RNA Preparation
by Jennifer Hornstein - 05/14/12
A number of RNA preparation technologies are widely available that can be classified into four general techniques: organic extraction methods, spin basket formats, magnetic particle methods, and direct lysis methods. While all can be used to prepare high-quality RNA suitable for a wide variety of analysis techniques, there are several factors to consider in selecting the right purification technology.
In addition to the information below, an RNA basics webinar is also available.
Is the sample particularly difficult to manage?
Tissues that are high in nucleases or fatty tissues, and samples with high amounts of inhibitors, can present particular problems.
How much sample do you need to process?
Larger sample sizes require kits that contain scalable chemistries. Generally, the larger the sample, the lower the throughput.
What throughput is required?
Some formats are particularly well suited to higher levels of throughput and automation.
Organic extraction methods of RNA preparation
These methods are considered the gold standard for RNA preparation. During this process, the sample is homogenized in a phenol-containing solution and the sample is then centrifuged. During centrifugation, the sample separates into three phases: a lower organic phase, a middle phase that contains denatured proteins and gDNA, and an upper aqueous phase that contains RNA. The upper aqueous phase is recovered and RNA is collected by alcohol precipitation and rehydration.
Benefits of organic RNA extraction
1. Rapid denaturation of nucleases and stabilization of RNA
2. Scalable format
Drawbacks of organic RNA extraction
1. The use and associated waste of chlorinated organic reagents
2. Laborious and manually intensive processing
3. Difficult to automate
Filter-based, spin basket formats - utilize membranes (usually glass fiber, derivitized silica, or ion exchange membranes) that are seated at the bottom of a small plastic basket. Samples are lysed in a buffer that contains RNase inhibitors (usually guanidine salts), and nucleic acids are bound to the membrane by passing the lysate through the membrane using centrifugal force. Wash solutions are subsequently passed through the membrane and discarded. An appropriate elution solution is applied and the sample is collected into a tube by centrifugation. Some formats can be processed by either centrifugation or vacuum using specialized manifolds. Hybrid methods that combine the effectiveness of organic extraction with the ease of sample collection, washing, and elution of spin basket formats also exist.
Benefits of spin basket formats
1. Convenience and ease of use
2. Amenable to single-sample and 96-well processing
3. Ability to automate
4. Ability to manufacture membranes of various dimensions
Drawbacks of spin basket formats
1. Propensity to clog with particulate material
2. Retention of large nucleic acids such as gDNA
3. Fixed binding capacity within a manufactured format
4. When automated, requirements for complex vacuum systems or centrifugation
Magnetic particle RNA preparation methods utilize small (0.5–1 µm) particles that contain a paramagnetic core and surrounding shell modified to bind to entities of interest. Paramagnetic particles migrate when exposed to a magnetic field, but retain minimal magnetic memory once the field is removed. This allows the particles to interact with molecules of interest based on their surface modifications, be collected rapidly using an external magnetic field, and then be resuspended easily once the field is removed. Samples are lysed in a solution containing RNase inhibitors and allowed to bind to magnetic particles. The magnetic particles and associated cargo are collected by applying a magnetic field. After several rounds of release, resuspension in wash solutions, and recapture, the RNA is released into an elution solution and the particles are removed.
Benefits of magnetic particle–mediated purification
1. No risk of filter clogging
2. Solution-based binding kinetics increase the efficiency of target capture
3. The magnetic format allows for rapid collection/concentration of sample
4. Increased ease of implementation on instrument platforms
5. Ability to automate
6. Wide availability of surface chemistries
Drawbacks of magnetic particles
1. Potential carry-through of magnetic particles into eluted samples
2. Slow migration of magnetic particles in viscous solutions
3. Capture/release of particles can be laborious when performed manually
Direct lysis methods - perform sample preparation (not purification) by utilizing lysis buffer formulations that disrupt samples, stabilize nucleic acids, and are compatible with downstream analysis. Typically, a sample is mixed with lysis agent, incubated for some amount of time under specified conditions, and then used directly for downstream analysis. If desired, samples can often be purified from stabilized lysates. By eliminating the need to bind and elute from solid surfaces, direct lysis methods can avoid bias and recovery efficiency effects that may occur when using other purification methods.
Benefits of direct lysis RNA preparation methods
1. Extremely fast and easy
2. Highest potential for accurate RNA representation
3. Can work well with very small samples
4. Amenable to simple automation
Drawbacks of direct lysis RNA preparation methods
1. Inability to perform traditional analytical methods such as spectrophotometric measurement of yield
2. Dilution-based (most useful with concentrated samples)
3. Potential for suboptimal performance unless developed/optimized with downstream analysis
4. Potential for residual RNase activity if lysates are not handled properly
Ambion® RNA isolation kits provides flexibility for sample size, type, and processing format, and includes kits for the isolation of total or poly(A) RNA. For additional information on approximately how much total or poly(A) RNA can be recovered from a given amount of tissue or cells, please refer to the technical information that accompanies each kit, or contact Life Technologies Technical Support.