Methods of extracting RNA
AssigneeNexGen Diagnostics LLC
InventorHashem Akhavan-Tafti
Life of this patent
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Abstract
Methods and materials are disclosed for rapid and simple extraction and isolation of RNA from a biological sample involving the use of an acidic solution and a solid phase binding material that has the ability to liberate nucleic acids from biological samples, including whole blood, without first performing any preliminary lysis to disrupt cells or viruses. No detergents or chaotropic substances for lysing cells or viruses are needed or used. Viral, bacterial and mammalian genomic RNA can be isolated using the method of the invention. RNA isolated by the present method is suitable for use in downstream processes such as RT-PCR.
Claims35
A method for extracting ribonucleic acid from a biological sample containing at least one of cells or viruses comprising:
- a) contacting the sample with an acidic solution to form a mixture;
- b) combining the mixture with a solid phase binding material selected to have the ability to liberate ribonucleic acid directly from biological samples without first performing any preliminary lysis, and wherein no chaotropic agents or detergents are used to effect lysis, and whereby the solid phase binding material causes lysis of cells and viruses to liberate ribonucleic acid;
- and
- c) binding ribonucleic acid on the solid phase.
A method for extracting ribonucleic acid from a biological sample selected from bacterial cultures, pelleted cells from bacterial cultures, blood, blood plasma, blood serum, urine sputum, semen, CSF, plant cells, animal cells, and tissue homogenates, the sample containing at least one of cells or viruses comprising:
- a) contacting the sample with an acidic solution having a pH in the range of 1-5 wherein the acidic solution comprises an aqueous solution of an organic or inorganic acid selected from pyridinium salts, mineral acids, monocarboxylic acids, dicarboxylic acids, tricarboxylic acids, and amino acids, and their alkali metal, alkaline earth, zinc, NH4+, quaternary ammonium and quaternary phosphonium salts to form a mixture;
- b) combining the mixture with a solid phase binding material comprising a matrix portion and a nucleic acid binding portion wherein the solid phase binding material is selected to have the ability to liberate ribonucleic acid directly from biological samples without first performing any preliminary lysis, and wherein no chaotropic agents or detergents are used to effect lysis, and whereby the nucleic acid binding groups cause lysis of cells and viruses to liberate ribonucleic acid;
- and
- c) binding ribonucleic acid on the solid phase.
A method for isolating ribonucleic acid from a biological sample selected from bacterial cultures, pelleted cells from bacterial cultures, blood, blood plasma, blood serum, urine sputum, semen, CSF, plant cells, animal cells, and tissue homogenates, the sample containing at least one of cells or viruses comprising:
- a) contacting the sample with an acidic solution having a pH in the range of 1-5, wherein the acidic solution comprises an aqueous solution of an organic or inorganic acid selected from pyridinium salts, mineral acids, monocarboxylic acids, dicarboxylic acids, tricarboxylic acids, and amino acids, and their alkali metal, alkaline earth, transition metal, NH4+, quaternary ammonium and quaternary phosphonium salts to form a mixture;
- b) combining the mixture with a solid phase binding material comprising magnetic particles having a tributylphosphonium nucleic acid binding group linked through a cleavable arylthioester linkage to a magnetic particle matrix wherein the solid phase binding material is selected to have the ability to liberate ribonucleic acid directly from biological samples without first performing any preliminary lysis, and wherein no chaotropic agents or detergents are used to effect lysis, and whereby the nucleic acid binding groups cause lysis of cells and viruses to liberate ribonucleic acid;
- and
- c) binding ribonucleic acid on the solid phase;
- d) separating the sample from the solid phase having ribonucleic acid bound thereto;
- e) optionally washing the solid phase with at least one wash solution;
- and
- f) cleaving the selectively cleavable linkage with a cleavage reagent thereby releasing the ribonucleic acid from the solid phase binding material.
Dependencies are inferred from the claim text.