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  Apparatus and system having dry control gene silencing compositionsUSPTO Application #: 20060166234Title: Apparatus and system having dry control gene silencing compositions Abstract: An RTF testing plate can include at least a first control well including a substantially dry first control composition having at least a first control siRNA. The first control siRNA is capable of providing a first indication of the gene silencing efficacy. Additionally, the first control composition can be configured such that the first control siRNA is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the first control well. The control siRNA can be any one of a transfection control siRNA, positive control siRNA, or negative control siRNA. Optionally, the total amount of control siRNA in the first control composition can be present in an amount for transfecting cells in only the first control well. (end of abstract) Agent: Jonathan M. Benns, Ph.d. Workman Nydegger - Salt Lake City, UT, US Inventors: Barbara Robertson, Devin Leake, Kathryn Robinson, William S. Marshall, Anastasia Khvorova USPTO Applicaton #: 20060166234 - 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 20060166234. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This United States patent application claims benefit of U.S. Provisional Application Ser. No. 60/630,320, filed Nov. 22, 2004, and U.S. Provisional Application Ser. No. 60/678,165, filed May 04, 2005, both of which are incorporated herein by reference. [0002] This United States Patent Application also cross-references the following United States patent applications filed herewith: Attorney Docket No. 16542.1.1, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING COMPOSITIONS, with Barbara Robertson, Ph.D., et al. as inventors; Attorney Docket No. 16542.1.2, entitled APPARATUS AND SYSTEM HAVING DRY GENE SILENCING POOLS, with Barbara Robertson, Ph.D., et al as inventors; and Attorney Docket No. 16542.1.4, entitled METHOD OF DETERMINING A CELLULAR RESPONSE TO A BIOLOGICAL AGENT, with Barbara Robertson, Ph.D., et al. as inventors, wherein each is incorporated herein by reference. BACKGROUND OF THE INVENTION [0003] 1. The Field of the Invention [0004] The present invention relates to an apparatus and system for use in RNA interference. More particularly, the present invention relates to an apparatus and system that include a well plate having control siRNA. [0005] 2. The Related Technology [0006] Recently, a natural cellular regulatory pathway was discovered that uses transcribed microRNA ("miRNA") in order to control protein production. The miRNA includes a duplex region of sense and antisense RNA. This regulatory pathway uses miRNA in order to target complementary mRNA to inhibit production of the encoded protein. Accordingly, a complex series of proteins are involved in this RNA interfering pathway to inhibit or stop production of the proteins encoded by the mRNA. As such, the process is referred to as RNA interference or RNAi. [0007] Additionally, it has been found that the RNAi pathway can be used with synthetic dsRNA (e.g., siRNA) for silencing genes and inhibiting protein expression. This can allow for siRNA having specific sequences to be produced to target complementary DNA and/or mRNA encoding a specific protein. The siRNA can interact with the natural RNAi pathway to silence a target gene and inhibit production of the encoded polypeptide. The ability to silence a specific gene and inhibit production of the encoded protein has been used for basic research of gene function, gene mapping, cellular pathway analysis, and other gene-related studies. [0008] In order to induce gene silencing, the siRNA needs to be introduced into a cell. While the most common procedures for introducing nucleic acids into cells has been forward transfection, reverse transfection ("RTF") has been developed more recently and used as an alternative to forward transfection procedures. In certain versions of RTF protocols, a complex of lipid-nucleic acid (e.g., lipoplex) can be prepared and introduced into the test wells of a well plate. Cells are introduced into the test wells with the lipid-nucleic acid complexes, and incubated so that the siRNA can enter the cells. Examples of some RTF protocols can be found in U.S. Pat. No. 5,811,274 to Palsson, U.S. Pat. No. 5,804,431 to Palsson and U.S. Pat. No. 6,544,790 to Sabatini and in U.S. Published Applications 2002/0006664 to Sabatini and 2003/070642 to Caldwell et al. As described in these references, RTF procedures for nucleic acids generally can have fewer steps compared to traditional forward transfection and may offer benefits in attempting to isolate the transfected cells to particular regions of a single surface, such as a glass slide. However, RTF procedures for siRNA have not been optimized to the point of practical application, and improvements in gene silencing efficacy are still needed, especially for situations in which one is experimenting with multiple different siRNAs, different gene targets or different cell lines. [0009] Often, RTF protocols can be performed on well plates in a manner that is not optimized or produces inaccurate and unreliable data. The lack of optimization can cause variations between plates, and can reduce the reliability of the data. Variations in data from a lack of optimization or an error can produce results that appear to be related to the gene silencing obtained from the siRNA, and are difficult to detect without comparing the test results to other experiments performed with the same siRNA. Additionally, systematic variations in data that occur can arise from the RTF conditions, and may be related to the amount of siRNA, amount of siRNA carrier, cell density, media, temperature, or various other factors that affect gene silencing. [0010] Therefore, it would be advantageous to have an improved RTF protocol for testing the efficacy of gene silencing. Additionally, it would be beneficial to have an RTF format that uses controls to test the efficacy of gene silencing. BRIEF SUMMARY OF THE INVENTION [0011] Generally, embodiments of the present invention include well plates, kits, systems, and methods of using the same for testing the efficacy of gene silencing. Accordingly, the present invention provides well plates, kits, and systems that implement an improved RTF testing protocol for delivering control siRNA into cells to test the efficacy of gene silencing in other cells in the well plate or other well plates. The control siRNA can provide an indication of gene silencing efficacy that can be compared to known functionalities and standard results obtained from using the control siRNA in optimal or other test conditions. [0012] In one embodiment, the present invention can include a reverse transfection plate for testing the efficacy of gene silencing. The plate can include at least a first control well including a substantially dry first control composition having at least a first control siRNA. The first control siRNA can be capable of providing a first indication of the gene silencing efficacy. Additionally, the first control composition can be configured such that the first control siRNA is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the first control well. The control siRNA can be any one of a transfection control siRNA, positive control siRNA, or negative control siRNA. Optionally, the total amount of control siRNA in the first control composition can be present in an amount for transfecting cells in only the first control well. [0013] In one embodiment, the plate can further include at least a second control well including a substantially dry second control composition. The second control composition can include at least a second control siRNA, which can be any of the transfection, positive, or negative control siRNAs. Preferably, the second control siRNA is different from the first control siRNA. As such, the second control siRNA can provide a second indication of gene silencing efficacy that is different from the first indication. The second control composition can be configured such that the second control siRNA is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the second control well. Optionally, the first control composition includes a positive control siRNA and the second control composition includes a negative control siRNA. Alternatively, the first control composition includes a positive control siRNA and the second control composition includes a transfection control siRNA. In yet another alternative, the first control composition can have a transfection control siRNA and the second control composition can include a negative control siRNA. [0014] In one embodiment, the plate can further include at least a third control well including a substantially dry third control composition. The third control composition can include at least a third control siRNA, which can be any of the transfection, positive, or negative control siRNA. Preferably, the third control siRNA is different, from the first control siRNA and second control siRNA. As such, the third control siRNA can provide a third indication of gene silencing efficacy that is different from the first indication and second indication. The third control composition can be configured such that the third control siRNA is capable of being solubilized or suspended in an aqueous medium in an amount sufficient for transfecting cells in the third control well. Preferably, the first, second, and third control siRNAs include a transfection, positive, and negative control siRNA, respectively. [0015] In one embodiment, the transfection control siRNA, positive control siRNA, or negative control siRNA can conform to the descriptions provided herein and in the incorporated references. [0016] In one embodiment, the present invention provides a kit or system that includes a well plate having a control well with a substantially dry control composition comprised of control siRNA. Additionally, such a kit or system includes a polynucleotide carrier. The polynucleotide carrier can be a cationic lipid, polymer, lipopolymer, or the like. Additionally, the kit or system can include various solubilizing solutions, reagents, cell culture media, and the like. [0017] In one embodiment, the present invention includes a method of testing the efficacy of gene silencing with control siRNA. This can include testing the conditions used in the RTF protocol, which may be related to cell density, type of polynucleotide carrier, carrier concentration, siRNA concentration, RTF protocol, or other factors that can alter the effectiveness for siRNA to silence genes. Additionally, such a testing method can include the use of any well plate consistent with the foregoing characterizations. Accordingly, an aqueous medium can be added to a first control well in the well plate, wherein the first control well includes a first control siRNA. The aqueous medium can solubilize or suspend the first control siRNA. Optionally, the aqueous medium can include a polynucleotide carrier such as a cationic lipid, polymer, lipopolymer, and the like, which can form a complex with the control siRNA. The complex can be prepared so as to be capable of being suspended or solubilized in the aqueous medium. [0018] Cells can be added to the first well under conditions that permit transfection with the complex. The cells can be added in an amount of about 1.times.10.sup.3 to about 3.5.times.10.sup.4 or about 2.times.10.sup.3 to about 3.times.10.sup.4 cells per about 0.3 cm.sup.2 to about 0.35 cm.sup.2 of cell growth surface area. Subsequently, the control siRNA can contact the cell in a manner that allows for entry into the cellular cytoplasm. However, any mode of transfection can be used to cause the control siRNA to enter the cell. The well plate can then be maintained under conditions so that cell growth, cell division, transfection, and/or gene silencing occurs. After a proper duration that allows for transfection and/or the control siRNA to silence a known gene, the effect of the first control siRNA on the cells can be determined. The effect of the first control siRNA in the cells can be compared with a known effect of the first control siRNA. [0019] In one embodiment, a second control siRNA can be used to test the efficacy of gene silencing. As such, the testing protocol can include adding the aqueous medium to a second control well in the well plate, wherein the second control well includes a second control siRNA. Subsequently, the cells can be added to the second control well under conditions that permit transfection, and the second control siRNA can then be transfected into the cells by any mode of transfection. The effect of the second control siRNA on the cells can be determined. The effect of the second control siRNA can be compared to the effect of the first control siRNA. [0020] In one embodiment, a third control siRNA can be used to test the efficacy of gene silencing. As such, the testing protocol can include adding the aqueous medium to a third control well in the well plate, wherein the third control well includes a third control siRNA. Subsequently, the cells can be added to the third control well under conditions that permit transfection, and the third control siRNA can then be transfected into the cells by any mode of transfection. The effect of the third control siRNA on the cells can be determined. The effect of the third control siRNA can be compared to the effect of the first control siRNA and/or the effect of the second control siRNA. [0021] In one embodiment, a blank well that is substantially devoid of siRNA can be used to test the efficacy of gene silencing. As such, the testing protocol can include adding an aqueous medium to a blank well in the well plate, the blank well being devoid of siRNA. Optionally, the aqueous medium can include the polynucleotide carrier. Optionally, the cells are then added to the blank well, and the effects of control siRNA on the cells are compared to the cells added to the blank well. On the other hand, the cells may not be added to the blank well so that it can be used for various calibrations. Continue reading... Full patent description for Apparatus and system having dry control gene silencing compositions Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Apparatus and system having dry control gene silencing compositions patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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