Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are present in all cells. DNA is the genetic substance of the cell, on which the genes can be transcribed into messenger RNA (mRNA), and the corresponding genetic information carried by the latter will be translated into a protein with functional activity. In addition to mRNA, there are other RNAs, including transfer RNA (tRNA), ribosomal RNA (rRNA), interference RNA (iRNA), micro RNA (miRNA), and heterologous nuclear RNA (hnRNA). Analyzing gene expression and regulation through assessment of mRNA, iRNA and miRNA in tissues or cells is an extremely important method in the field of life science, and provides a very large number of physiological indicators for cells. A wide range of techniques can be used to assess mRNA, iRNA and miRNA level, including polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), real-time PCR (Real-Time PCR), Northern blotting, bio-chip, and transcriptome sequencing, etc. When using each of these techniques, it is necessary to use the isolated RNA samples, and the RNA samples should not contain the following contaminant found in active cells: genomic DNA, protein, fat, polyphenols, polysaccharides and certain other biological molecules. [Wyatt, J. R, and Tinoco, I. J. (1993) RNA structure and RNA function, in The RNA World (Gesteland, R. F and Atkins, J. F., eds.), Cold Spring Harbore Laboratory Press, Cold Spring Harbor, N.Y., pp. 465-496.]
To separate RNA from a kind of biological material, RNA must be separated from DNA and other ingredients constituting the biological specimens. The key of RNA purification is: 1. All enzymes that have nuclease activity, including nucleic acid enzymes (RNases), must be deactivated to prevent decomposition of RNA by these enzymes. Peel off the attached protein on the natural RNA to release the bare RNA to the solution; this can prevent the RNA from being removed together with the attached protein as impurities, resulting in increased RNA extraction yield. At the same time, it also helps prevent RNA decomposition caused by residual trace protein in the obtained RNA samples, which improves the quality and purity of the isolated RNA. The obtained RNA samples must not contain any protein residue, because these protein residues usually contain enzymes with RNases activity, which will lead to RNA decomposition.
In addition to nuclease, many other enzymes in the cell also have RNase activity, such as the variety of DNA polymerases, transcription enzymes, reverse transcriptase enzymes with RNase activity that are present in eukaryotic and prokaryotic cells. Therefore, the key to preventing RNA decomposition is completely inhibiting the activity of all enzymes, including RNases, prior to RNA extraction. Thus, in the RNA samples obtained, even trace protein must be removed since it may contain some enzymes with RNase activity, which may cause the RNA obtained to be quickly decomposed.
Many scientists invented and expounded a variety of RNA isolation method. Most of these methods depend on cesium chloride density gradient centrifugation or phenol chloroform extraction. However, the isolation process is time-consuming, and uses hazardous reagents that pose threat to the environment and human body. In addition, the samples are easily contaminated by exogenous nucleic acid and protein. [U.S. Pat. Nos. 5,075,430; 5,234,809; 5,155,018; 6,277,648; 6,875,857; 6,958,392; 6,953,686; 6,310,199; 6,992,182; 6,475,388; 5,075,430; 7,074,916; U.S. Patent Publication No. 20060024701; European Patent No. EP0765335; Boom et al. 1990, J. Clinical Microbiology 28: 495; Cox, R. A. (1968) The use of guanidinium chloride in the isolation of nucleic acids Methods Enzymol 12, Part B, 120-129; Chirgwin, J, M., Przybyka, A. E., Mac Donald, R. J., Rutter, W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18(24), 5294-5299]
In early RNA extraction experiments, usually phenol and chloroform were used for protein extraction [Kirby, K S (1968) Isolation of Nucleic Acids with Phenolic Solutions, Methods Enzymol 12, part B, 87-99.]; With this method, a large amount of RNA molecule for which the protein is not completely peeled off will be removed as impurities together with the proteins removed; furthermore, some RNA in the obtained RNA samples still contain a small amount of protein, so repeated phenol and chloroform extraction is required to remove the residual protein. This results in a very low yield of extracted RNA, and the entire RNA extraction process is complicated and lengthy, and the trace protein that's not completely removed often causes the extracted RNA to be decomposed. [Ingle, J. and Burns, R. G. (1968) The loss of ribosomal ribonucleic acid during the preparation of nucleic acid from certain plant tissues by the detergent-pjenol method, Biochem J. 110, 605, 606]
In order to address the various defects of the phenol and chloroform extraction method used to extract RNA, cesium chloride gradient centrifugation method was used to selectively precipitate RNA to obtain high-purity nucleic acid, which provides a good alternative to phenol and chloroform extraction. However, this method requires very expensive equipment, strict operational training and complicated operation, which inhibits its application in general-purpose laboratories and cannot be widely adopted. [Glisin, V., Crkvenjakov, R., and Byus, C (1974) Ribonucleic and isolated by cesium chloride centrifugation. Biochemistry 13, 2633-2637.]
Currently the most widely used method is the technique invented by P. Chomczynski (U.S. Pat. No. 5,945,515), which he expounded in his published thesis. [Chomczynski, P. and Sacchi, N, (1987) Single-step method of RNA isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biocemistry 18, 5294-5299; and Sacchi, N, (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Bilchem 162(1), 156-159.]
In his patent, P. Chomczynski discloses a solution that enables simultaneous isolation of RNA, DNA and protein. The solution contains guanidine thiocyanate, 40 to 60% of phenol, glycerin used as phenol stabilizer, and the buffer used to maintain a solution pH of 4. The solution is a homogeneous mixture (i.e., single-phase liquid), and addition of 10% chloroform may lead to effective phase-split and enable RNA to be assigned to the aqueous phase, and the protein and DNA are concentrated in between the organic phase and two phases. Adding an equal volume of isopropanol to the aqueous phase results in RNA precipitation. The RNA precipitate obtained through centrifugation can be washed using 70% ethanol and dried.
Compared with the traditional technique, the advantage of this method is the time needed is less than 1 hour and the operation intensity is also greatly reduced. It provides high product purity and high efficiency. However, due to the following problems, it can only be used in the laboratory, and cannot be widely adopted:
It requires expensive laboratory facilities and supplies, such as high-speed refrigerated centrifuge, liquid nitrogen, low-temperature refrigerator, a fume hood.
It requires skilled technical staff. To use this method, you need trained and highly specialized personnel with rich experience in experimental work.
The supplies used, such as centrifuge tubes, micro pipette tip, glassware and reagents, require DEPC water treatment or high-temperature, long-time baking to prevent RNA decomposition by the intractable RNases. This requires a lot of time and effort.
The method not only uses highly toxic phenol and chloroform, but uses high-concentration (2˜5M) guanidine salt solution with acute toxicity, which are extremely harmful to human health—guanidine thiocyanate and guanidine hydrochloride are defined as hazardous materials by CHIP (Commonwealth Chemicals Hazard Information and Packaging) and are defined as toxic reagents by HCS (United States Hazard Communication Standard).
E. Although the combined effect of high-concentration guanidinium salts, phenol and chloroform in peeling protein off from RNA is greatly improved, the protein cannot be completely removed, which often leads to decreased yield of RNA extracted from some biological materials, and since a small amount of residual protein is present in the RNA sample obtained, the Rnases contained in the residual protein can cause RNA decomposition.
F. Furthermore, for tissues that do not dissolve in guanidine salt, particularly most tissues in plants, the isolation method using high-concentration guanidine salt does not produce any RNA.
USPTO Backgrounds
USPTO Backgrounds