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  Methods and compositions for the rapid isolation of small rna moleculesUSPTO Application #: 20070202511Title: Methods and compositions for the rapid isolation of small rna molecules Abstract: The present invention provides extraction compositions and methods for the rapid and efficient isolation of small RNA molecules from a biological sample. In particular, the extraction compositions, when contacted with a biological sample, releases the small RNA molecules from the other molecules in a biological sample, and the released small RNA molecules may then be isolated. (end of abstract) Agent: Polsinelli Shalton Welte Suelthaus PC - St. Louis, MO, US Inventors: Fuqiang Chen, Carol Kreader USPTO Applicaton #: 20070202511 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070202511. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to methods, compositions, and kits to isolate small RNA molecules from biological samples. BACKGROUND OF THE INVENTION [0002] More than a decade ago a non-coding 22-nucleotide (nt) RNA (lin-4) was discovered that played an important role in the developmental timing of Caenorhabditis elegans. It was not realized, however, until just a just few years ago that small RNA molecules such as lin-4 are ubiquitous and play important regulatory roles in virtually all eukaryotes. Recent work has shown that prokaryotes and viruses also express small regulatory RNA molecules. Thus, in addition to large RNA molecules, such as messenger RNA (mRNA) and ribosomal RNA (rRNA), cells express an array of small RNA molecules, including 5.8S rRNA, 5S rRNA, transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA); micro RNA (miRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), small temporary RNA (stRNA), tiny non-coding RNA (tncRNA), small scan RNA (scRNA), and small modulatory RNA (smRNA). Micro RNA molecules, which are processed from larger primary transcripts and range from 20-23 nucleotides in length, have emerged as a hot topic in molecular biology research because of their important roles in a wide range of biological processes, including gene regulation, cell differentiation, growth, and development, as well as certain disease states. Other small RNA molecules, such as siRNAs, are also involved in gene silencing and genome modification. [0003] The long delay to the realization of the existence and importance of small RNA could, in part, be attributed to the fact that small RNA molecules are often unintentionally eliminated because of their small sizes from preparations of natural RNA populations. Furthermore, small RNA molecules represent a very small fraction in terms of weight of the total RNA population, and without removal of abundant RNAs and enrichment of small RNAs, their detection could be severely hampered. Historically, variations of two methods have been used to isolate RNA from biological samples. The first method relies on chemical extraction with organic solvents such as phenol and chloroform under acidic conditions to separate DNA and other biomolecules from the RNA, which is then concentrated by alcohol precipitation. Alcohol precipitation, however, does not quantitatively recover small RNA molecules. The second method relies on immobilization of RNA on a solid support binding matrix, such as silica. For this, the RNA-containing sample is mixed with a high salt solution or a salt and alcohol mixture to decrease the affinity of RNA for water and increase its affinity for the silica matrix. Small RNA, however, binds poorly to the support matrix under the conditions routinely used. Thus, most existing RNA preparation methods and commercial RNA purification kits are deficient in capturing small RNA. [0004] With the recent surge of interest in miRNA and other small RNA molecules, the standard isolation procedures have been modified to facilitate the isolation of small RNA. These methods largely rely on phenol and chloroform extraction and step-wise alcohol fractionation. For example, U.S. Publication No. 2005/0059024 discloses a method in which a cell lysate is extracted with phenol and chloroform to partition the genomic DNA into an interphase between an organic lower phase and an aqueous upper phase. The aqueous upper phase is collected and mixed with a low percentage of alcohol and applied to a first binding matrix. The large RNA is immobilized onto the first matrix and the small RNA flow through the matrix. The flow-through fraction is then mixed with a higher percentage of alcohol and applied to a second binding matrix, to which the small RNA binds and can be recovered. Thus, small RNA can be isolated and purified using a multi-step procedure. A major drawback of the current methodology is the use of phenol and chloroform, not only because they pose potential health hazards but also because they are ineffective with certain biological material, such as plant tissues that are rich in phenolic or polyphenolic compounds. Another drawback of the current methodology is that phase separation and alcohol fractionation are laborious and time consuming, making them incompatible with high throughput and automation demands. [0005] The present invention provides methods and compositions for the rapid isolation of small RNA from a variety of biological sources without using phenol and chloroform extraction or alcohol gradient fractionation. SUMMARY OF THE INVENTION [0006] The present invention encompasses compositions and methods to rapidly and efficiently isolate small RNA molecules from a biological sample. One aspect of the invention is an extraction composition comprising a chaotropic agent and a metal salt. [0007] Another aspect of the invention provides a method for isolating small RNA from a biological sample. In the method, the biological sample is contacted with a chaotropic agent and a metal salt. After contact, small RNA is released from debris in the biological sample. The small RNA remains in solution, allowing it to be separated from the debris by a variety of methods known in the art. [0008] A further aspect of the invention encompasses a kit comprising solutions to prepare an extraction composition, all concomitant agents and buffers, means to isolate the small RNA, and complete instructions. [0009] Other aspects and features of the invention will be in part apparent and in part described in more detail herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0010] It has been discovered that contacting a biological sample with a chaotropic agent and a metal salt leads to the release of small RNA from other biomolecules. In particular, contact with the chaotropic agent and metal salt selectively precipitates the large RNA, genomic DNA, and other large macromolecules, whereas the small RNA remains in solution. The small RNA may be readily separated and isolated from the aggregated macromolecules. As illustrated in the examples, the methods and compositions of the present invention allow the rapid isolation of pure preparations of small RNA in high yield from a variety of organisms, including, plant tissue, mammalian cultured cells, mammalian tissue, yeast cells, and bacterial cells. I. Extraction Compositions [0011] One aspect of the invention encompasses an extraction composition. Typically the extraction composition will have a chaotropic agent and a metal salt. In this context, the term "composition" is used in its broadest sense to mean use of a chaotropic agent and metal salt for the separation of small RNA from a biological sample. The term composition does not mean that the two agents have to be contacted with the biological sample at the same time as a part of the same solution. It is contemplated for example, as described below, that the chaotropic agent and metal salt may be contacted with the biological sample either simultaneously as part of the same mixture or added sequentially, one reagent after the other. As will be appreciated by a skilled artisan, the extraction composition may optionally include a variety of other agents without departing from the scope of the invention. Suitable non-limiting examples of agents comprising the extraction composition are detailed below. (a) Chaotropic Agent [0012] A variety of chaotropic agents are suitable for use in the extraction composition. Generally speaking, the chaotropic agent denatures proteins, disrupts membranes, releases nucleic acids, protects RNA from degradation, and facilitates cell lysis. Examples of suitable chaotropic agents include guanidine hydrochloride, guanidine thiocyanate, guanidine carbonate, sodium iodide, sodium perchlorate, sodium trichloroacetate, urea, and thiourea. The chaotropic agent may be incorporated into the extraction composition alone or as a combination of two or more chaotropic agents. As will be appreciated by one skilled in the art, the choice of chaotropic agent will be determined by the origin of material from which small RNA is to be isolated. In one embodiment, the chaotropic agent is guanidine thiocyanate. Guanidine thiocyanate, however, is not particularly suitable for RNA isolation from certain plant tissues, such as cotton leaves, grape leaves, red maple leaves, and gymnosperm conifer needles, which are rich in phenolic or polyphenolic compounds. In another embodiment, the chaotropic agent is a combination of two or more quanidinium salts. In a preferred embodiment, the chaotropic agent is guanidine hydrochloride. [0013] The concentration of the chaotropic agent or the combination of chaotropic agents in the extraction composition may and will vary but may range from about 1 M to about 8 M. Lower concentrations of a chaotropic agent may be used if cell disruption and RNase inhibition are not major concerns. In one aspect, the concentration of the chaotopic agent is about 3 M. In another aspect, the concentration of the chaotopic agent is about 6 M. In another aspect, the concentration of the chaotopic agent is about 4 M. In yet another aspect, the concentration of the chaotopic agent is about 5 M. (b) Metal Salt [0014] The extraction composition includes at least one metal salt. A variety of metal salts are suitable for use in the invention. The metal salt may be incorporated into the extraction composition before or after contacting the biological sample. The metal salt may be a group IA metal salt or a group IIA metal salt. Suitable examples of group IIA metals include beryllium, magnesium, calcium, strontium, and barium. Suitable examples of group IA metals include lithium, sodium, potassium cesium, and francium. In a preferred embodiment, the metal salt is a lithium salt. Examples of suitable lithium salts include lithium acetate, lithium borate, lithium carbonate, lithium chloride, and lithium citrate. In a preferred embodiment, the lithium salt is lithium chloride. [0015] The concentration of metal salt or combination of metal salts may range from about 1 M to about 8 M. In one aspect, the concentration of lithium salt ranges from about 1.5 M to about 6 M. In one embodiment, the concentration of lithium chloride is about 6 M. In another embodiment, the concentration of lithium chloride is about 2.4 M. In another embodiment, the concentration of lithium chloride is about 1.8 M. In yet another embodiment, the concentration of lithium chloride is about 3.6 M. [0016] Without being bound by any particular theory, it is believed that the combination of a chaotropic agent and a lithium salt in the extraction composition creates a discriminating environment that is particularly suitable for the separation of large RNA from small RNA. It is known that Li.sup.+ ions have a very high charge/radius ratio and a unique affinity for RNA molecules. They can effectively neutralize the negative charges on the RNA backbone and remove much of the water shell from the RNA molecule. A chaotrope, on the other hand, has a strong disrupting ability, which can keep the charge-neutralized RNA molecules from collapsing on each other and becoming aggregated. As a result of the counteraction, each charge-neutralized RNA molecule may behave as a discrete entity in the extraction composition. It is further believed that charge-neutralized large RNAs possess a higher density than the extraction composition and, therefore, they are very susceptible to precipitation, whereas charge-neutralized small RNAs have a lower density than the extraction composition and, therefore, they substantially remain in solution. The density of each RNA molecule may also be affected to some extent by pH, for H.sup.+ can compete with Li.sup.+ for the negative charges on the RNA backbone. As a consequence, the extraction composition is optimized for extracting small RNA, as detailed below. (3) pH and Buffer Continue reading... Full patent description for Methods and compositions for the rapid isolation of small rna molecules Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods and compositions for the rapid isolation of small rna molecules patent application. 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