RNA is an intermediate product of gene expression and exists in the cytoplasm and nucleus. RNA extraction is the basis of molecular biology research. High-quality, high-purity and high-integrity of is required for downstream molecular biology experiments such as cDNA library construction, in vitro reverse transcription, real-time fluorescence quantitative PCR, and Northern hybridization analysis. However, RNA is very “delicate”, and it will fail to be extracted if not carefully enough.
1. Introduction to RNA
RNA is the abbreviation of RiboNucleicAcid, which is a genetic information carrier existing in biological cells and some viruses and viroids. RNA is a long chain molecule formed by the condensation of ribonucleotides through phosphodiester bonds. Unlike DNA, RNA is generally a single-stranded long molecule. Using one DNA strand as a template and the principle of base complementary pairing, a single strand is formed by transcription. Its main function is to realize the expression of genetic information on proteins, and it is a bridge in the process of transforming genetic information to phenotype. Some organisms, such as SARS-CoV-2, also directly use RNA as a carrier of genetic information.
2. Principles of RNA extraction
The essence of total RNA extraction from different tissues is to lyse cells, release RNA, and remove impurities such as polysaccharides, phenols, proteins, and DNA in different ways, and finally obtain high-purity RNA products through a series of extraction, washing and precipitation. There are four main principles for RNA extraction: 1. Ensure the integrity of the primary structure of RNA; 2. There should be no organic solvents that inhibit the enzyme and high concentrations of metal ions in the extracted RNA samples; 3. The contamination of other biological macromolecules such as proteins, polysaccharides and lipid molecules should be minimized; 4. The contamination of other nucleic acid molecules (DNA) should be excluded.
3. The evolution of RNA extraction technology
With the in-depth research and wide application of RNA, RNA extraction technology has also been greatly developed, from the widely used traditional manual RNA extraction to automated RNA extraction by nucleic acid extractors.
( 1 ) Traditional manual RNA extraction technology
Early RNA extraction techniques
The early RNA extraction technology was guanidine isothiocyanate cesium chloride ultracentrifugation, the principle of which was to use the protein denaturing agent guanidine isothiocyanate to effectively inhibit the activity of RNase. Density gradient ultracentrifugation in cesium chloride medium precipitates RNA at the bottom of the tube. However, this extraction method is complicated in operation, long in process, limited in the number of samples extracted at one time, and requires high experimental equipment. Later, in order to solve the problem of lack of scientific research equipment such as ultracentrifugation, scientists proposed the guanidine hydrochloride-organic solvent method, which uses guanidine salts to inhibit RNase, homogenize cells to lyse, extract proteins with organic solvents, and remove DNA by selectively precipitating RNA molecules. , Although this method solves the problem of no ultracentrifuge, the whole operation process is still complicated and time-consuming.
RNA extraction technology by Trizol method
With the development of science and technology, there have been many techniques for manually extracting and purifying RNA, the most classic of which is the Trizol method. The main components of Trizol reagent are phenol and guanidine isothiocyanate. The main function of phenol is to lyse cells, so that the protein and nucleic acid substances in the cells are depolymerized and released. Although phenol can effectively denature proteins, it cannot completely inhibit RNase activity. Guanidine isothiocyanate is a powerful protein denaturing agent, which can not only dissolve proteins rapidly, resulting in the fragmentation of cell structures, and the rapid separation of nucleoproteins from nucleic acids due to the destruction and disappearance of their secondary structures, but also has a strong denaturing effect on RNases. When chloroform is added, it can extract the acidic phenol, which can promote the RNA into the aqueous phase, and after centrifugation can form the aqueous and organic layers, so that the RNA is separated from the protein and DNA remaining in the organic phase. The aqueous layer (colorless) is mainly RNA, and the organic layer (yellow) is mainly DNA and protein. Trizol reagent can be used not only for small samples, but also for a large number of samples. It is suitable for animal, plant, bacteria, blood extraction, and can process a large number of different samples at the same time.
Column extraction and magnetic bead extraction techniques
Although the Trizol method is widely used and the quality of the RNA obtained is also good, the Trizol reagent itself is harmful to human beings. Therefore, new techniques for manual RNA extraction have emerged one after another, such as silica gel column extraction and manual magnetic bead method. Among them, the column extraction kit adopts a unique lysis system, which does not require toxic phenol/chloroform extraction, and can rapidly lyse tissue or cells at the same time inhibit the endogenous RNase released by the cells, maintain the integrity of the RNA, and obtain RNA through the adsorption column. The principle of the manual magnetic bead method is that specific active functional groups with adsorption on the surface of the magnetic beads can adsorb nucleic acids specifically and efficiently. After the cells are lysed, magnetic beads are added to adsorb nucleic acids, and impurities are removed by washing reagent purification to obtain pure nucleic acids. However, manual RNA extraction technology has always been time-consuming, cumbersome, and inefficient.
( 2 ) Automated RNA extraction technology
In recent years, molecular diagnostic technology has developed rapidly, and the operation of experiments is developing in a simpler and more humanized direction. There are dozens of sample types for molecular diagnostic testing, and the detection items applicable to the processed RNA products also cover various molecular biology technology platforms such as fluorescence quantitative PCR, gene sequencing, and gene chips. However, no matter what kind of test item it is, accurate test results undoubtedly depend on high-quality specimens and specimen pre-processing. The magnetic bead method RNA extraction reagent plus the automatic nucleic acid extraction instrument have become the perfect combination to solve the pretreatment of clinical molecular diagnostic samples.
4. Manual extraction of blood RNA samples vs Automatic extraction
Blood consists of two parts: plasma and blood cells. Blood RNA is mainly found in white blood cells, and red blood cells are very rich in ribonuclease (RNase), which will cause serious degradation of RNA. Minimizing the effects of RNase is the key to ensuring high-quality RNA extraction from blood.
Compared with manual extraction, automated extraction of blood RNA is simpler and faster, which can effectively reduce the operational errors of manual extraction and minimize the contamination of exogenous RNases to ensure the quality and purity of the extracted RNA.
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