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Reagent for the isolation of RNA

a technology of rna and reagents, applied in the field of reagents, can solve the problems of low rna yield of commercial reagents and kits available for the isolation of rna, poor rna quality, and inability to accommodate difficult specimens, and achieve the effect of high quality

Inactive Publication Date: 2005-07-21
INVITROGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides reagents, methods, and kits for extracting RNA from difficult materials such as plants, especially those containing phenolics, tannins, polysaccharides, and resins. The present invention can achieve higher yields of RNA compared to conventional methods and the RNA preparations obtained have high quality as demonstrated by superior A260 / 280 results and gel electrophoresis.

Problems solved by technology

Commercial reagents and kits available for the isolation of RNA do not accommodate difficult specimens, especially those specimens rich in polyphenolics (e.g., conifer needles) or starch (e.g., potato tuber or seeds).
RNA yields from these reagents and kits are low, or RNA quality is poor as demonstrated by low A260 / 280 ratios or gel electrophoresis.
However, all of these known methods are extremely laborious.

Method used

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  • Reagent for the isolation of RNA

Examples

Experimental program
Comparison scheme
Effect test

example 1

Small Scale Protocol RNA Isolation with Chloroform Extraction

[0039] Fresh tissue, e.g., plant leaf or root, was ground to a powder in liquid nitrogen. Dried seed was ground at room temperature. All ground plant material was stored at −70° C. To 0.1 g of ground tissue was added 0.5 ml of the present RNA Isolation Reagent (e.g., 20% 2-mercaptoethanol formulation). The sample was mixed until the ground tissue was thoroughly re-suspended, and then let stand for 5 minutes at room temperature.

[0040] The sample was centrifuged for 2 minutes at 12,000×g in a microcentrifuge. The supernatant was transferred to an RNase-free tube. A 0.1 ml aliquot of 5M NaCl was added to the supernatant and the sample was mixed. An aliquot of 0.3 ml of chloroform was added and mixed. The sample was centrifuged at 4° C. for 10 minutes at 12,000×g to separate the phases. The aqueous phase was transferred to an RNase-free tube, and an equal volume of isopropyl alcohol was added. The sample was mixed and let st...

example 2

Small Scale Protocol with RNA Cartridge Purification

[0041] Fresh tissue, e.g., plant leaf or root, was ground to a powder in liquid nitrogen. Dried seed was ground at room temperature. All ground plant material was stored at −70° C. To 0.1 g of ground tissue was added 0.5 ml of the present RNA Isolation Reagent (e.g., 20% 2-mercaptoethanol formulation). The sample was mixed until the ground tissue was thoroughly re-suspended, and then let stand for 5 minutes at room temperature.

[0042] The sample was poured onto a Concert Homogenizer and centrifuged for 2 minutes at 12,000×g in a microcentrifuge to clarify the RNA extract. To the flowthrough was added an equal volume of guanidinium isothiocyanate and ethanol, and processed through the Concert RNA cartridge, washed, and the RNA was eluted with water, according to the protocol provided by the manufacturer.

example 3

Large Scale Protocol for Isolating RNA from Plants

[0043] Fresh tissue was ground to a powder in liquid nitrogen. Dried seed was ground at room temperature. All ground plant material was stored at −70° C. To 1 g of ground tissue was added 5 ml of the present RNA Isolation Reagent (e.g., 20% 2-mercaptoethanol formulation), mixed until the sample was thoroughly re-suspended, and let stand for 5 minutes at room temperature. The sample was centrifuged at 4° C. for 5 minutes at 2600×g in a tabletop centrifuge. The supernatant was transferred to an RNase-free tube, passing the solution through a 100-μm nylon sieve. A 1 ml aliquot of 5M NaCl was added to the supernatant, and 3 ml of chloroform, and mixed. The sample was centrifuged at 4° C. for 30 minutes at 2600×g to separate the phases. The aqueous phase was transferred to an RNase-free tube, and an equal volume of isopropyl alcohol was added. The sample was mixed and let stand at room temperature for 10 minutes. The sample was centrifug...

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Abstract

The present invention provides RNA extraction reagents, methods and kits that are especially useful for extracting RNA. The reagents, methods and kits of the present invention are especially useful for extracting RNA, for example, cytoplasmic RNA, from difficult materials, from plants, especially, difficult plant tissues, such as those containing phenolics, tannins, polysaccharides (such as starch) and resins. Comparative high yields are obtainable according to the present invention when compared to conventional reagents and methods. The RNA preparations obtained in accordance with the present invention are also of high quality as demonstrated by superior A260 / 280 results and by gel electrophoresis.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The invention is a reagent, methods and kits for the isolation of RNA from RNA containing cells and tissues, and preferably from cells and / or tissues from plants and plant materials. [0003] 2. Related Art [0004] Commercial reagents and kits available for the isolation of RNA do not accommodate difficult specimens, especially those specimens rich in polyphenolics (e.g., conifer needles) or starch (e.g., potato tuber or seeds). RNA yields from these reagents and kits are low, or RNA quality is poor as demonstrated by low A260 / 280 ratios or gel electrophoresis. [0005] Several methods are described in the literature for the isolation of RNA from pine needles and spruce needles that are reported to give RNA of good quality. See, e.g., Schneiderbauer, A. et al, Isolation of Functional RNA from Plants Rich in Phenolic Compounds Analytical Biochemistry 197: 91-95 (1991); Graham, Glenn C., A method of extraction of total RNA...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/02C12N15/10C12N15/09
CPCC12N15/1003
Inventor SIMMS, DOMENICA
Owner INVITROGEN
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