RNA extraction (Microorganism, blood, cell lines, Tissues)

RNA extraction, along with DNA and protein extraction, is a fundamental technique in molecular biology. These biomarkers can be isolated from biological samples for various research, diagnostic, and therapeutic purposes. RNA extraction involves the separation and purification of RNA from different biological samples, including body tissues, cells, body fluids, blood, and more. The quality and quantity of the extracted RNA are crucial factors to ensure the accuracy of gene expression analysis and other laboratory applications in molecular research. The presence of ribonuclease (RNase) in tissues and cells is the biggest obstacle to RNA extraction, as this enzyme rapidly degrades single-stranded RNA.

How RNA Extraction is Performed

DNA extraction methods cannot be used for RNA extraction because RNA is structurally very different from DNA. RNA is a single-stranded biomolecule, whereas DNA is double-stranded. Additionally, RNA is more sensitive than DNA to environmental factors such as temperature and ribonucleases; therefore, RNA extraction must be performed under special conditions.

The protocol for extracting RNA from various biological samples is generally the same, but there are slight differences in the protocol based on the type of target sample. For example, extracting RNA from tissue involves more steps compared to extracting from cellular pellets, as several preparation steps must be performed on the tissue sample. Another example of these differences is the extraction of RNA from neuronal cells and brain tissue, which is rich in lipids, making high-quality RNA extraction difficult.

To obtain high-quality RNA with a high degree of purity, several factors must be considered: the final RNA must be free from any contamination, including proteins, genomic DNA, and inhibitors of cDNA synthesis and DNA polymerases, which include phenol, chloroform, and ethanol. Additionally, the absence of nucleases in the extracted RNA is critical to prevent RNA degradation. PCR reactions and other molecular techniques are significantly affected by the RNA extraction process and the presence of various contaminants such as hemoglobin, fats, calcium ions, etc. Therefore, setting up the RNA extraction protocol from cells and tissues is crucial for obtaining acceptable results.

Types of RNA Extraction Methods

Three primary techniques are commonly used for RNA extraction:

Phenol-Chloroform Extraction

Silica-Membrane Based Spin Column Technology

Magnetic Particle Extraction

Phenol-Chloroform Extraction

The liquid-liquid extraction technique based on phenol-chloroform is the most common method for RNA extraction from tissues and cells, and it is the basis for most extraction kits used. This method has advantages and disadvantages. The biggest drawback of this method is contamination with cellular components and other solvents. Additionally, this method requires standard protective conditions in the laboratory, such as access to a chemical hood. The main advantage of this method is the extraction of high-quality and high-quantity RNA, and it can be easily set up.

Silica-Membrane Based Spin Column Technology

The silica column-based RNA extraction technique does not require toxic solvents, and the level of contamination from other components in the target sample is lower in RNA extracted using this method. The spin columns for nucleic acid purification contain silica resin that binds to RNA. Under low pH conditions, RNA molecules specifically bind to the silica membrane, while proteins and polysaccharides pass through. Impurities are then washed away, and finally, under low-salt conditions, the RNA is eluted from the membrane for storage.

Magnetic Particle Extraction

Magnetic RNA isolation is a simple and efficient method currently in use. Many commercial magnetic carriers are available in kits. Magnetic carriers contain ligands made from biopolymers that have an affinity for the target nucleic acids used in the isolation process. RNA extraction using magnetic beads is performed on various samples, particularly viruses, bacteria, and blood. The magnetic beads are very small, and their surfaces are coated with charged materials that can bind to RNA.

When the magnetic beads are added to the desired biological sample, the lysed cellular components are attracted towards the poles when passing through a magnetic field, thereby separating other cell components from the RNA. The RNA bound to the magnetic beads is then placed in salt buffers to elute the RNA from the beads. Although the purity percentage of RNA extracted using this technique is high, it is an expensive method and has not yet become a routine laboratory procedure.

Conclusion

High-quality RNA extraction is the first and most fundamental step for performing many molecular techniques such as Reverse Transcription Real-Time PCR (RT-qPCR), transcriptome analysis using sequencing, array analysis, Northern blotting, and cDNA synthesis. In addition to the RNA extraction protocol, various factors affect the quality of this biomarker, including the quality of the biological sample, the materials used, and the method of storing the extracted RNA. Besides the correct selection of the extraction method and the setup of the procedure, some important considerations must be taken into account during RNA extraction to ensure that the RNA obtained is acceptable in terms of both quantity and quality.