Methods of RNA extraction
The process of isolating RNA from biological samples is called RNA extraction. This procedure is challenging due to ubiquitous RNases presenting in cells and tissues, leading to RNA’s rapid degradation. There are three major techniques extensively used for RNA extraction:
1. Organic extraction methods
These methods are considered as the golden standard for the isolation of total RNA. During this procedure, the sample is homogenized in the phenol-Guanidine Isothiocyanate (GITC)-based solutions and then centrifuged. The sample is separated into three phases; the upper a bottom organic phase, a middle phase containing denatured proteins, the upper aqueous phase that contains RNA and DNA. By altering the pH to less than 7.0, DNA is easily denatured and precipitates into the middle phase, leaving the RNA in the aqueous phase. Finally, the upper phase is separated, and RNA is collected via alcohol precipitation, particularly isopropyl alcohol.
Advantages: Rapid denaturing of nucleases and stabilization of RNA
Disadvantages: Laborious and manually intensive procedure, being toxic
2. Silica-membrane based spin column technology
Spin column-based RNA purification is a solid phase extraction method that contains a silica resin binding selectively to RNA and quickly purifies it. This method relies on the salt setting and other specific conditions. This approach uses chaotropic salts that denature proteins (including DNases and RNases) and denature nucleic acids by disrupting their hydrogen bonding.
The method includes the cell lysis, the binding nucleic acid to silica gel membrane, washing the bounded nucleic acid to the silica-gel membrane, and eluting the nucleic acid. The lysis procedure is undertaken by breaking the cell membrane with lysis enzymes. A binding buffer is then added to a spin column, and the column is put in a centrifuge. The centrifuge forces the binding solution through a silica gel membrane that is inside the spin column. In the optimal pH and salt concentration, the desired nucleic acid will bind to the silica-gel membrane. This step is followed by adding ethanol or isopropanol to remove the salt. The washing buffer is then added to the column and put into the centrifuge, passing the wash buffer through the membrane. This way, remaining contaminants will exit from the membrane, resulting in the purified RNA bound to the silica gel. After removing the washing buffer, RNA is eluted from the column with an elution buffer (low-salt solution) or only water. Finally, the elution buffer removes the RNA from the membrane, and it is collected from the bottom of the column.
Advantages: Simple, fast, and relatively non-toxic high-yielding method for RNA extraction
Disadvantages: Not applicable for high-throughput processing and challenging to automate, laborious, and manually intensive procedure.
3. Paramagnetic particle technology
This approach uses small particles (0.5- 1 μm) containing paramagnetic cores that are being stationary. These tiny particles apply a magnetic field through a sample volume, resulting in the physical separation of both the magnetic beads and bound analytes from the solution. Samples are lysed in the buffer containing RNase inhibitors and incubated with the magnetic beads, allowing the particles to bind RNA molecules. After separation, the fluid phase is removed, and the following washing solutions are added. Since the paramagnetic particles retain minimal magnetic memory after removing the magnetic field, they can repeat the separation process when mixing the beads into the new solution. Finally, magnetic particles are collected by applying a magnetic field.
Advantages: Being quick, inexpensive, having efficient purification and easy automation, voiding the cross-contamination, and isolating RNA from the larger sample volumes.
Disadvantages: No control over field strength, impeding the migration of magnetic beads in case of viscous samples, being laborious and manually intensive procedure, and having the risk of RNA sample’s contamination with residual magnetic beads.
