Methods for RNA isolation :

1. Organic Extraction Methods

Organic extraction 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 extraction

 Rapid denaturation of nucleases and stabilization of RNA

 Scalable format

Drawbacks of organic extraction

 The use and associated waste of chlorinated organic reagents

 Laborious and manually intensive processing
 Difficult to automate .
2. Magnetic Particle Methods

Magnetic particle 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 :

  No risk of filter clogging
 Solution-based binding kinetics increase the efficiency of target capture
 The magnetic format allows for rapid collection/concentration of sample
 Increased ease of implementation on instrument platforms
 Ability to automate
 Wide availability of surface chemistries

Drawbacks of magnetic particles :

 Potential carry-through of magnetic particles into eluted samples

 Slow migration of magnetic particles in viscous solutions
 Capture/release of particles can be laborious when performed manually

3. Direct Lysis Methods

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 methods

 Extremely fast and easy

 Highest potential for accurate RNA representation
 Can work well with very small samples
 Amenable to simple automation
 Scalable

Drawbacks of direct lysis methods

 Inability to perform traditional analytical methods such as spectrophotometric

measurement of yield
 Dilution-based (most useful with concentrated samples)
 Potential for suboptimal performance unless developed/optimized with downstream
analysis
 Potential for residual RNase activity if lysates are not handled properl