This application is a continuation application of U.S. application Ser. No. 10/354,727 filed Jan. 30, 2003, which is a continuation of U.S. application Ser. No. 09/771,256 filed Jan. 26, 2001, now U.S. Pat. No. 6,528,641 issued Mar. 4, 2003, which is a continuation of PCT Application No. PCT/US99/17375 filed Jul. 30, 1999, which claims priority to and is a continuation-in-part of U.S. patent application Ser. No. 09/127,435 filed Jul. 31, 1998, now U.S. Pat. No. 6,204,375 issued Mar. 20, 2001. The entire disclosure of each of the above-referenced applications is incorporated herein by reference in its entirety.
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OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of molecular biology and provides a novel method and reagent for preserving and protecting the ribonucleic acid (RNA) content of tissue or cell samples from degradation prior to RNA isolation.
2. Description of Related Art
Obtaining high quality, intact RNA is the first and often the most critical step in performing many fundamental molecular biology experiments. Intact RNA is required for quantitative and qualitative analysis of RNA expression by Northern blot hybridization, nuclease protection assays, and RT-PCR.
There are many published reports which describe methods to isolate intact RNA from fresh (or quick frozen) cells or tissues. Most of these techniques utilize a rapid cell disruption step in which the tissue is dispersed in a powerful protein denaturation solution containing a chaotropic agent (e.g., guanidinium or lithium salt). This rapid disruption of cell membranes and inactivation of endogenous ribonuclease is critical to prevent the RNA from being degraded.
To obtain high quality RNA it is necessary to minimize the activity of RNase liberated during cell lysis and to prevent RNA degradation from other sources. This is normally accomplished by using isolation methods that disrupt tissues and inactivate or inhibit RNases simultaneously. For specimens low in endogenous ribonuclease, isolation protocols commonly use extraction buffers containing detergents to solubilize membranes, and inhibitors of RNase such as placental ribonuclease inhibitor or vanadyl-ribonucleoside complexes. RNA isolation from more challenging samples, such as intact tissues or cells high in endogenous ribonuclease, requires a more aggressive approach. In these cases, the tissue or cells are quickly homogenized in a powerful protein denaturant (usually guanidinium isothiocyanate), to irreversibly inactivate nucleases and solubilize cell membranes. If a tissue sample can not be promptly homogenized, it must be rapidly frozen by immersion in liquid nitrogen, and stored at −80° C. Samples frozen in this manner must never be thawed prior to RNA isolation or the RNA will be rapidly degraded by RNase liberated during the cell lysis that occurs during freezing. The tissue must be immersed in a pool of liquid nitrogen and ground to a fine powder using mortar and pestle. Once powdered, the still-frozen tissue is homogenized in RNA extraction buffer. In the laboratory, quick freezing of samples in order to delay RNA extraction carries the penalty of a substantial increase in hands-on processing time. Processing multiple samples with liquid nitrogen and mortar and pestle is extremely laborious.
Quick freezing is even less convenient outside of the laboratory environment, but is still considered a necessity by those in the field. Scientists in the field collecting specimens for analysis do not have access to a high-speed homogenizer. They are forced to carry a supply of liquid nitrogen or dry ice large enough to store samples until they can be transferred to an ultra-low temperature freezer. Similarly, RNA extracted from human biopsy samples is usually partly or mostly degraded because pathologists do not routinely flash freeze specimens to preserve RNA.
There have been attempts to isolate RNA from archival samples that have not been prepared by the flash-freezing methodology. For example, Esser et al., 1995 claim the isolation of full length RNA from cells fixed with 5% acetic acid, 95% ethanol, with RNase inhibitors. However, in this paper, isolated cells in suspension were fixed in acetic acid/ethanol solution at −20 ° C. and then held at 4° C. for a relatively short time. Unfortunately, testing by the Inventor has shown that the Esser et al. 95% ethanol/5% acetic acid solution does not meet the performance standards required by the present invention. RNA recovered from both tissue samples and spleen cells in suspension kept at 4° C. for 20 hours appeared partially degraded, while RNA isolated from tissues stored at ambient temperature was completely degraded. Experiments reported in Esser et al. show that the method results in loss of RNA, due to leakage from the cells caused by ethanol. Using that method, 70% of the RNA is lost immediately upon fixation, and after 1 hour, 80% of the RNA is gone. Further, in a test where tissue samples and spleen cells were stored in the 95% ethanol/5% acetic acid solution at 25° C. overnight, the RNA of both the cell and tissue samples was completely degraded. Data is shown in FIG. 1.
The use of high purity, intact RNA is fundamental for performing various molecular biological assays and experiments such as Northern blot hybridization, nuclease protection assays, RT-PCR and medical diagnosis. The intrinsic instability of RNA and the presence of RNases in samples makes the isolation of intact RNA a difficult procedure. Further, the isolation and assay of RNA-containing samples is typically time consuming and tedious. The contamination of a molecular biology laboratory with RNases due to human error can have catastrophic results. Thus, there is an ongoing need to develop improved techniques, to make RNA isolation and assay methods more sensitive, more specific, faster, easier to use and less susceptible to human error and handling. It would therefore be advantageous in many instances, for research facilities to use automated RNA preservation protocol. For example, the present invention, could be combined with rapid RNA assay techniques or integrated nucleic acid diagnostic devices (U.S. Pat. No. 5,726,012, U.S. Pat. No. 5,922,591, incorporated by reference) for efficient, automated RNA preservation and analysis.
U.S. Pat. No. 5,256,571 reports a cell preservative solution comprising a water-miscible alcohol in an amount sufficient to fix mammalian cells, an anti-clumping agent and a buffering agent. At least one paper, Dimulescu et al., reports the apparent use of this fixative to preserve cervical cancer cells and cord blood lymphocytes prior to RNA isolation.
A large body of literature suggests that ethanol and acetone combinations are the best known fixatives for future recovery of nucleic acids from archival tissue. Yet, in view of the studies of the inventors, such ethanol/acetone mixture does not provide all of the desired characteristics of an RNA preservation medium. The mixtures do not protect RNA at ambient temperature, does not allow for the preservation of RNA in solid, multi-cell samples, and are also flammable, which makes it intrinsically less attractive as a general use reagent.
Some peripherally related art exists that deals with aspects of preserving or recovering RNA from fixed or preserved tissue samples. These reports include numerous evaluations of the suitability of histological fixatives to maximize the signal obtained by in situ hybridization to detect (not recover) RNA in tissue samples (for example U.S. Pat. Nos. 5,196,182 and 5,260,048). Other reports detail methods to recover fragmented RNA from fixed tissues for limited molecular analysis by PCR™ (Koopman et al., Foss et al., Stanta et al., Houze et al.). To recover this fragmented RNA, samples are typically treated with proteinase K to degrade the structural components of the tissue, then the RNA is extracted with a guanidinium-based solution. The RNA recovered from fixed tissue is of extremely poor quality, averaging in size of about 200 bases (Stanta 1991). This is probably due to a number of factors including the action of endogenous RNase and cross-linking of the RNA in the intracellular matrix during fixation. Since the RNA is mostly degraded, it can not be used for northern analysis or nuclease protection assays. It can be used in RT-PCR, but only for amplification of very small fragments.
The use of ammonium sulfate to precipitate proteins out of solution is known, but the use of ammonium sulfate to preserve RNA does not, to the Inventor's knowledge, appear in the art. Two reports describe the use of ammonium sulfate to investigate the folding and activity of mammalian ribonuclease A (Allewell et al. and Lin et al.). Allewell et al. investigated the effects of ammonium sulfate on the folding and activity of RNase A. At pH 5.5, the activity of ribonuclease A is suppressed to approximately 10% of the untreated control level across a broad range of ammonium sulfate concentration. This suppression of activity was expected by the authors. It appears to be due to a salt-induced denaturation of the protein. Unfortunately, even 10% RNase activity would substantially degrade the RNA of a sample over time. Therefore, this inhibition is not sufficient to protect RNA in many applications. When the ammonium sulfate is at pH 7.0, the activity of RNase A is suppressed at low concentrations as expected, but unexpectedly rises to 110% of the level of the untreated control at higher concentrations (3M). The authors theorize that the combination of the neutral pH and the high salt concentration forces a refolding of the protein into an alternate, highly active configuration. However, the Allewell et al. group were examining the activity of pure RNase A in solution, rather than in a cellular sample containing many RNases.
In view of the above, there is a need for methods and reagents that allow one to preserve and recover high quality, intact RNA from tissue samples stored at near ambient or ambient temperature.
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OF THE INVENTION
The present invention relates to a novel method and reagents for preserving the RNA in tissue fragments at temperatures above the freezing point of the preservatives for extended periods of time, including days to months, prior to RNA isolation. There is no prior report disclosing any reagent or method similar to that described in this application. This breakthrough alleviates the necessity of either immediately processing samples to extract RNA, or the restriction of only isolating tissue at sites which have a supply of liquid nitrogen or dry ice.
The present application relates to compositions of RNA preservation media and methods of preserving RNA comprising: (1) obtaining an RNA-containing sample; and (2) treating the sample with an RNA preservation medium that infiltrates the sample, and protects the RNA from nucleases. In a preferred embodiment, the RNA preservation medium brings about the precipitation the RNA in the sample along with cellular protein in the sample. This co-precipitation of the RNA and cellular proteins is believed to render the RNA inaccessible to nucleases via physical means, while the action of the RNA preservation medium simultaneously inactivates or inhibits the action of the nucleases.
In some preferred embodiments, the RNA preservation medium comprises a salt that precipitates the RNA in the sample along with the cellular protein. In more presently preferred embodiments, the salt is a sulfate salt, for example, ammonium sulfate, ammonium bisulfate, cesium sulfate, cadmium sulfate, cesium iron (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate, lithium sulfate, magnesium sulfate, manganese sulfate, potassium sulfate, sodium sulfate, or zinc sulfate. In presently preferred commercial embodiments, the salt is ammonium sulfate.
In RNA preservation media comprising salt, the salt is typically present in a concentration sufficient to precipitate the RNA in the sample along with the cellular protein. The salt is typically present in a concentration between 20 g/100 ml and the saturating concentration of the salt. Specifically, salt concentrations of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 g/100 ml may be used, and the concentration may be a range defined between any two of these concentrations.
Of course, during use, some dilution of the salt concentration may occur due to, for example, liquid in the sample. Therefore, these salt concentrations may be higher than the final salt concentrations obtained in some uses. Further, it is contemplated that amounts of salt above the saturating concentration may be used in regard to the present invention. In such embodiments, there may be salt that is not in solution in the RNA preservation medium. This should not affect the RNA preservation abilities of the media. In fact, media having more than a saturating concentration of a salt may have some utility in applications where the media is added to a liquid sample. In such cases, upon addition to the liquid sample, salt which is not in solution prior to addition, may become soluble due to the increase in liquid volume. Thus, the final concentration of a salt can be at a level higher than that possible if a preservation medium containing a saturating or less than saturating salt concentration were used.
Preferred salts that have a solubility of greater than 20 g/100 ml are: ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate, magnesium chloride, manganese sulfate, manganese chloride, potassium chloride, potassium sulfate, sodium chloride, sodium acetate, sodium sulfate, zinc chloride, zinc acetate, or zinc sulfate.
In a preferred embodiment, the salt is ammonium chloride at a concentration of between 20 g/100 ml and 100 g/100 ml, 30 g/100 ml and 100 g/100 ml, or 30 g/100 ml and 80 g/100 ml. In a currently preferred commercial embodiment, the salt is ammonium sulfate is in a concentration of 70 g/100 ml.
The present invention is not limited to the use of ammonium sulfate, and other salts or compounds will also be useful in protecting RNA in tissue samples and cell samples, for reasons as follows. The solubility of individual proteins depends greatly on the pH and salt concentration of the aqueous environment. Virtually all proteins are insoluble in pure water. As the ionic strength of the medium increases, proteins become more soluble. This is known as “salting in” of proteins. Above some ionic strength, the solubility of protein decreases. The precise condition at which this occurs is unique for each protein/salt combination. In fact, at some salt concentrations, one protein may be completely insoluble while another is at its solubility maximum. This phenomenon is known as “salting out.” Some salts have a much more dramatic salting out effect at high concentrations than others (e.g., NO3<Cl−<acetate−<SO42−). This phenomenon is the consequence of certain inherited characteristics of the ions (e.g., size, hydration, size, etc.). The present RNA protection media are believed to function due to the salting out effect of high levels of salt. The theory is that the salting out of proteins in the cells of the tissue samples or cell samples is what leads to the formation of RNA-protective protein/RNA complexes. The importance of the “salting out” phenomenon to this application is several-fold. First, it highlights that the effectiveness of RNA protection by protein precipitation using high concentrations of salt is complex and that certain combinations of ionic strength (salt concentration) and pH may make a particular salt much more effective in one formulation than at a different pH or concentration. Second, it provides a firm scientific foundation for the basic mechanism of action by the reagents in this application, and guides one in the search for additional RNA protective compounds within the scope of the invention. In order to determine whether another salt or putative RNA protective compound will function in the methods and reagents of the invention, one need merely obtain that salt or compound and test it in the manners described in the examples. By following the teachings of the examples, one of skill can easily elucidate whether a candidate substance is actually an RNA protective compound. Third, based on the theory that precipitation of intracellular proteins is the key to protecting RNA in situ, this explains why alcohol and acetone (agents that also can precipitate protein, albeit by a different mechanism) are partially active at protecting RNA in tissues, albeit not as protective as needed for most applications.
In some embodiments, the RNA preservation medium will comprise a combination of at least two salts that precipitate the RNA in the sample along with the cellular protein. In this manner, it might be that the total concentration of any given salt will not exceed 20 g/100 ml. However, in anticipated preferred embodiments, the combination of at least two salts is present in a total salt concentration sufficient to precipitate the RNA in the sample along with the cellular protein. In some embodiments, the total salt concentration is between 20 g/100 ml and 100 g/100 ml.
The RNA preservation medium may further comprise ethanol, methanol, acetone, trichloroacetic acid, 1-propanol, 2-propanol, polyethylene glycol, or acetic acid. These additional potential components can precipitate proteins in preserved cells and thereby protect RNA. However, these additional potential components are not salts. It is anticipated that in some embodiments, one will use these organic solvents in combination with a concentration of salt to obtain one of the inventive RNA preservation media described herein. For example, it is anticipated that a combination of one or more of these organic solvents in conjunction with less than 20 g/100 ml salt will accomplish the goals of the invention.
In some embodiments, the RNA preservation medium comprises a salt such as ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate, magnesium chloride, manganese sulfate, manganese chloride, potassium chloride, potassium sulfate, sodium chloride, sodium acetate, sodium sulfate, zinc chloride, zinc acetate, zinc sulfate, methanol, trichloroacetic acid, 1-propanol, 2-propanol, polyethylene glycol, or acetic acid. Further, the RNA preservation medium may comprise a chelator of divalent cations, for example EDTA.
Typically, the RNA preservation medium comprises a buffer so that a constant pH can be maintained. For example, the buffer can be sodium citrate, sodium acetate, potassium citrate, or potassium acetate. In a presently preferred commercial embodiment, the buffer is sodium acetate. Typically, the RNA preservation medium has a pH of between 4 and 8. In presently preferred commercial embodiments, the pH is 5.2.
The sample preserved in the RNA preservation media may be any of a number of types of samples. For example, the sample may be a suspension of cells, such as bone marrow aspirates, white blood cells, sperm, blood, serum, plasma, bacteria, tissue culture cells, or algae. Alternatively, the sample may a solid tissue, for example, the tissue sample is from brain, heart, liver, spleen, thymus, kidney, testis, ovary, tumors, tissue biopsies, plant stems, roots, or leaves. In some cases, the sample may comprise an entire organism. For example, the organism may be a fish, insect, tadpole, coral, or embryo. In some protocols, it will be of benefit to hold an organism or sample in the RNA preservation medium during dissection. For example, it might be of benefit to dissect an organism in the RNA preservation medium when the sample comprises an organism that is a pathogen within a tissue sample or other organism. In this manner, the RNA of the pathogen may preserved. Further, the RNA of the tissue sample or other organism is preserved.
In many preferred methods, the practice of the invention will further comprise the step of isolating the preserved RNA. One of the advantages of the present RNA preservation media is that the RNA may be isolated from the tissue at a higher temperature than allowed with previous techniques. For example, the RNA may be isolated at a temperature that is greater than −20° C. In fact, the RNA may be isolated at room temperature.
In some cases, the sample may be stored in the RNA preservation medium prior to the isolation of the RNA. For example, the tissue is stored unfrozen at −20° C. to 45° C. Owing to the salt content of some of the RNA preservation media, samples are not frozen at −20° C. In preferred embodiments the sample may be stored at greater than 0° C.
The invention also contemplates kits for preserving RNA within a sample and isolating the RNA from the sample comprising: (1) an RNA preservation medium that infiltrates the sample and protects or partitions the RNA from nucleases; and (2) a reagent for performing an RNA extraction from the sample. In some embodiments, the reagent for performing an RNA extraction is a reagent for performing RNA extraction without organic solvents. Further, the reagent for performing an RNA extraction may be a reagent for performing a guanidinium-based RNA extraction. Alternatively, the reagent for performing an RNA extraction is a reagent for performing a lithium chloride-based RNA extraction.
In other embodiments of the present invention, a method of preserving RNA will comprise obtaining an RNA-containing sample, providing a salt and admixing the sample and the salt in a liquid to form an RNA preservation composition that infiltrates the sample and protects the RNA from nucleases. In one embodiment, the sample is comprised in the liquid prior to admixing the sample with the salt. In another embodiment, the salt is in a solid form prior to admixing with the sample and the liquid. In yet another embodiment, the salt is comprised in the liquid prior to admixing the sample with the salt.
In one embodiment of the invention, the sample is a blood cell and the liquid is blood serum. In another embodiment the sample is urine. In other embodiments, the liquid is water. In yet other embodiments, the liquid is a buffer.
In certain embodiments, RNA preservation compositions comprise obtaining an RNA-containing sample, providing a salt and admixing the sample and the salt in a liquid. The liquid can be a component of the sample, or added to the sample and salt. The salt is typically present in a concentration sufficient to precipitate the RNA in the sample along with the cellular protein. The salt is typically added so as to result in a final concentration in solution between 20 g/100 ml and the saturating concentration of the salt. In some cases, it will be efficient to add a very concentrated salt, a saturated salt or a supersaturated salt to a liquid sample. Adding salt that is likely to result in a greater than saturating concentration of salt in the final liquid sample has the advantages of quickly allowing RNA preserving concentrations to be reached and avoiding the need to carefully consider the amount of salt needed to reach a specific concentration in a given sample. Further, any salt that does not go into solution, will not affect the RNA preserving properties of the composition. Specifically, final salt concentrations of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 g/100 ml may be used, and the concentration may be a range defined between any two of these concentrations. In some cases, a liquid containing more than a saturating concentration of salt may be employed, with the expectation that additional volume of liquid in the sample will result in dissolution of salt concentration.