WO2010070657A1 - Composition pour l'élimination de couleurs et d'inhibiteurs de tissus de plante pour isoler de l'arn - Google Patents

Composition pour l'élimination de couleurs et d'inhibiteurs de tissus de plante pour isoler de l'arn Download PDF

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Publication number
WO2010070657A1
WO2010070657A1 PCT/IN2009/000612 IN2009000612W WO2010070657A1 WO 2010070657 A1 WO2010070657 A1 WO 2010070657A1 IN 2009000612 W IN2009000612 W IN 2009000612W WO 2010070657 A1 WO2010070657 A1 WO 2010070657A1
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WO
WIPO (PCT)
Prior art keywords
composition
rna
colours
removal
inhibitors
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Application number
PCT/IN2009/000612
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English (en)
Inventor
Ravi Shankar Singh
Sanjay Kumar
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Council Of Scientific & Industrial Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Council Of Scientific & Industrial Research filed Critical Council Of Scientific & Industrial Research
Publication of WO2010070657A1 publication Critical patent/WO2010070657A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • the present invention relates to a composition hereinafter referred to as colinmin for the removal of colours and inhibitors from plant tissues to isolate RNA.
  • this invention relates to a composition and the method to remove the soluble substances that otherwise impart colour to the ribonucleic acid (hereinafter known as RNA) preparations inhibiting down stream applications including synthesis of complementary DNA
  • cDNA polymerase chain reaction using RNA as one of the substrates.
  • soluble substance in the present invention is in context to the solution used to remove these substances.
  • RNA isolation is crucial to various molecular biology techniques involving RNA as starting material. These include reverse transcription-polymerase chain reaction (hereinafter, referred to as RT-PCR), real-time quantitative PCR, ⁇ croarrays, RNA mapping, in vitro translation, Northern blot analysis, nuclease protection assays and cDNA library construction.
  • RT-PCR reverse transcription-polymerase chain reaction
  • ⁇ croarrays RNA mapping
  • in vitro translation Northern blot analysis
  • nuclease protection assays and cDNA library construction.
  • RNA extraction (WO 2007/113614) developed a two-soluti ⁇ n system for rapid isolation of RNA composed of solution I [buffer saturated phenol (pH less than 7), SDS (0.1-1%), EDTA (10-2OmM), sodium acetate(0.3-0.8M)], and solution II [Dt' 'C(0.001-0.1%) treated deionized water].
  • This system has been referred to as iRIS system of RNA extraction in this disclosure.
  • RNA using various methods has been described in such cases such as RNeasy technology
  • the inventors of the present invention realized that there exists a need to develop a composition which will simply and quickly remove colours and interfering chemicals from plant tissues from which RNA is to be isolated.
  • the main object of the present invention is to provide a composition for removal of colours and inhibitors from plant tissues to isolate RNA.
  • Another object of the present invention is to provide a method for removal of colours and inhibitors from plant tissues to isolate RNA.
  • Another object of the present invention is to add aesthetic value to the isolated RNA by removing colours that sometimes is not liked by the researchers.
  • Yet another object of the present invention is to provide a kit for removal of colours and inhibitors from plant tissues to isolate RNA at an early stage thus making RNA suitable for downstream applications.
  • TRIzol reagent Invitrogen
  • iRIS system without the composition colinmin and method as claimed in the present invention failed to give good quality of RNA.
  • We assessed the quality of RNA by various methods such as absorbance spectrum analysis (using spectrophotometer), electrophoresing on denaturating agarose gel and RT-PCR based amplification of 26S rRNA.
  • composition colinmin and method as claimed in the present invention While with the composition colinmin and method as claimed in the present invention, the remarkable improvement in quality of RNA was observed with no co-precipitation of colour and inhibitory substances, lower absorption spectrum in visual range and undegraded (intact bands of) RNA on denaturating agarose gel.
  • the present invention provides a composition for the removal of colours and inhibitors from plant tissues comprising:
  • the invention further provides a process for the removal of colours and inhibitors from plant tissues to isolate RNA, characterized in that it employs the composition as claimed in claim 1, wherein the steps comprising: [a] grinding 10-100 mg of a plant tissue to a fine powder in liquid nitrogen;
  • step Cd centrifuging the thawed mixture as ' obtained in step Cd] at 13000 to 14000 rpm for 5 to 15 minutes at a temperature of 4 to 6 0 C and decanting the supernatant;
  • Figure 1 represents total RNA isolated from roots of Arnebia euchroma using various combinations of solutions as described in Example 1, in Panel (A) while Panel (B) shows RT-PCR (Reverse Transcription-Polymerase Chain Reaction) based amplification of 26S rRNA performed to assess the quality of RNA.
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • Figure 2 represents total RNA isolated from roots of Arnebia Vietnameseroma, Rheum australe and Daucus carota using TRIzol reagent (Invitrogen) without colinmin (A) and with colinmin (B) the claimed composition of the present invention colinmin following method as claimed in the present invention.
  • Figure 3 represents absorbance spectrum of RNA isolated from roots of Arnebia Vietnameseroma, Rheum australe and Daucus carota using TRIzol reagent (Invitrogen).
  • Figure 4 represents RT-PCR (Reverse Transcription-Polymerase Chain Reaction) based amplification of 26S rRNA using RNA isolated TRIzol reagent (Invitrogen) from roots of Arnebia Vietnameseroma, Rheum australe and Daucus carota.
  • Figure 5 represents total RNA isolated from roots of Arnebia Vietnameseroma, Rheum australe and Daucus carota using iRIS system without colinmin (A) and with colinmin (B) the claimed composition of the present invention colinmin following method as claimed in the present invention.
  • Figure 6 represents absorbance spectrum of RNA isolated from roots of Arnebia Vietnameseroma, Rheum australe and Daucus carota using iRIS system.
  • Figure 7 represents RT-PCR (Reverse Transcription-Polymerase Chain Reaction) based amplification of 26S rRNA using RNA isolated from roots of Arnebia Vietnameseroma, Rheum australe and Daucus carota; iRIS system without colinmin (A) and with colinmin (B) the claimed composition of the present invention colinmin following method as claimed in the present invention.
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • the present invention provides a composition named as colinmin and method for removal of colours and inhibitors from plant tissues to isolate RNA.
  • composition colinmin was prepared as follows: Deionised water used in the present invention was treated with 0.1% diethylpyrocarbonate (DEPC) in final concentration and autoclaved after leaving overnight at room temperature.
  • DEPC diethylpyrocarbonate
  • Sodium dodecyl sulphate used as an anionic detergent to inhibit the activity of RNAses.
  • Other salt substitutes for sodium dodecyl sulphate could be lithium salts ' of dodecyl sulfate as well as N-lauroyl sarcosine.
  • ethylene diamine tetraacetate (EDTA) used as a preferred chelating agent to reduce DNase activity.
  • Cyclhexane diamine tetraacetate (CDTA) could be the other substitute for EDTA.
  • RNA from tissues using the claimed composition Following steps comprised the method to isolate RNA from tissues using the claimed composition: 1. Grinding 10-100 mg of plant tissue to a fine powder in liquid nitrogen using a pestle and mortar.
  • step (8) Pellet thus obtained from step (8) can be processed further for RNA isolation following protocol such as iRIS system and TRIzol reagent (Invitrogen).
  • Protocol for RNA isolation using TRIzol Reagent (Invitrogen): 1. Plant Tissue (100 mg) was ground in liquid nitrogen to fine powder.
  • Plant tissue (100 mg) was ground in liquid nitrogen using mortar and pestle to make fine powder.
  • RNA pellet was washed with 70% ethanol, air-dried and dissolved in appropriate amount of DEPC treated water.
  • the solution I as used above comprises of buffer saturated phenol (pH less than 7), SDS(0.1-1%), EDTA(10-2OmM), sodium acetate(0.3-0.8M).
  • the solution II as used above comprises of diethylpyrocarbonate in deionised water having a conductivity of 17-18.2 mega-ohms.
  • the said tissue is preferably the roots of Arnebia Vietnameseroma, Rheum australe, and Daucus carota.
  • the anionic detergent is selected from the group consisting of dodecyl sulphate salt or N-lauroyl sarcosine.
  • the anionic detergent is preferably sodium dodecyl sulphate.
  • the chelating agent is selected from the group consisting of disodium and dipotassium of ethylene diamine tetra acetic acid.
  • the chelating agent is preferably EDTA.
  • the concentration of diethylpyrocarbonate used is about 0.1% volume /volume.
  • the present invention also provides a kit for the removal of colours and interfering chemicals from tissues comprising of: a) Colinmin and b) Instructions for using the composition.
  • RNA yield (ug/100mg tissue) using iRIS System and TRIzol Reagent (Invitrogen) with and without the composition of the present invention
  • Root tissues 100 Rheum Arnebia euchroma mg used for RNA australe Daucus carota isolation
  • Composition of present invention used for removal of colours and inhibitors from plant tissues comprises of 0.05-0.2 % sodium dodecyl sulphate, 8-15 mM EDTA and 70-90% ethanol in DEPC treated deionized water.
  • DEPC treated deionized water 50-200 mg sodium dodecyl sulphate, 8-15 mM EDTA (pH 8.0) and DEPC treated deionized water mixed together to make 25 ml of solution A and reconstituted with 75 ml of 95-98% ethanol just prior to use.
  • RNA quality and yield was found to be good with solution II, HI, VII and VIII.
  • RT-PCR based amplification of 26S rRNA ( Figure IB) performed to assess the quality of RNA isolated using different combination of claimed composition. RT-PCR based amplification found to be good with solution II, III, VII and VIII. We obtained best results in terms of quality and quantity with solution VII.
  • step (8) Pellet thus obtained from step (8) is further processed for RNA isolation.
  • RNA isolation protocols were followed for comparison of efficacy Protocol for RNA isolation using TRIzol Reagent (Invitrogen);
  • Plant Tissue (100 rag) was ground in liquid nitrogen to fine powder.
  • Plant tissue 100 mg was ground in liquid nitrogen using mortar and pestle to make fine powder. 2. Added 2 ml of solution I.
  • RNA pellet was washed with 70% ethanol, air-dried and dissolved in appropriate amount of DEPC treated water.
  • the solution I as used above comprises of buffer saturated phenol (pH less than 7), SDS(0.1-l%), EDTA(10-2OmM), sodium acetate(0.3-0.8M).
  • the solution II as used above comprises of diethylpyrocarbonate in deionised water having a conductivity of 17-18.2 mega-ohms.
  • RNA was quantified using spectrophotometer; the purity of RNA was determined by calculating the ratio of absorbance measured at 260 and 280 nm. In the present investigation, a value for the ratio between 1.8-2.0 was considered ideal for the purity of RNA. The quality of RNA isolated was assayed on 1.2% agarose gel containing formaldehyde.
  • RNA 5-6 ⁇ g of RNA mixed with 10 ⁇ l 2x RNA Loading dye (GenHunter, U.S.A.) and incubated at 65 0 C for 10 min and electrophoresed at 72 volts in IX MOPS buffer (60 mM sodium acetate, 2 mM MOPS and 0.1 mM EDTA), as described by Sambrook, J., Fritsch, E. F., Maniatis, T., (1989) ( In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY). Absorption spectrum of isolated RNA was recorded at wavelength ranging 380 to 780 nm using a spectrophotometer.
  • IX MOPS buffer 60 mM sodium acetate, 2 mM MOPS and 0.1 mM EDTA
  • RNA isolated using TRIzol reagent (Invitrogen) and iRIS system with the composition colinmin and method claimed in the present invention and method as described in Example 1-2 and Table 1 describes the yields obtained from different tissues.
  • RNA yield ( ⁇ g/lOOmg tissue), obtained from RNA isolation using iRIS System and TRIzol Reagent (Invitrogen) with and without the composition colinmin of the present invention and method as described in Example 1-3.
  • RNA yield obtained from roots of Arnebia Vietnameseroma, Rheum australe and Daucus carota as described in Table 1.
  • RT-PCR Reverse transcription-polymerase chain reaction
  • oligonucleotide primers specific to 26S rRNA were used to amplify this cDNA during 25 cycles, where a cycle was defined as 94° C for 30 seconds, 52° C for 40 seconds, and 72° C for 1 minute.
  • the composition of reaction mixture was 2.5 ⁇ ⁇ 1OX PCR buffer (20 mM Tris-HCl, pH 8.4, 50 Mm KCL, 1.5 mM MgCl 2 ), 0.5 ⁇ ⁇ Deoxynucleotide triphosphate (dNTPs,10 mM), 1.0 ⁇ 1 Forward primer (10 ⁇ M), 1.0 ⁇ l Reverse primer(10 ⁇ M), 1.0 ⁇ l cDNA, 0.25 ⁇ l Taq DNA polymerase (5U/ ⁇ L) and 18.75 ⁇ 1 sterile distilled water.
  • dNTPs Deoxynucleotide triphosphate
  • Present invention ensures removal of colours and inhibitors from plant tissues.
  • Present invention adds aesthetic value to the isolated RNA.
  • 3.Present invention shows compatibility with other RNA isolation methods that fail to yield good quality of RNA.

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  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Crystallography & Structural Chemistry (AREA)
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Abstract

La présente invention concerne une composition et un procédé pour l'élimination de couleurs et d'inhibiteurs de tissus pour isoler de l'ARN. En particulier, cette invention concerne une composition et le procédé pour éliminer les substances solubles qui sinon conféreraient une couleur à des préparations d'acide ribonucléique (ARN) ou inhiberaient des applications en aval comprenant la synthèse d'ADN complémentaire (ADNc) et la réaction de polymérase en chaîne en utilisant l'ARN en tant qu'un des substrats. Les protocoles rapportés à ce jour peuvent isoler l'ARN, mais l'ARN isolé n'est pas adapté pour des procédés en aval tels que la RT-PCR en raison de la présence de couleurs et des agents interférents associés. Aucun des protocoles ne concerne un procédé simple et rapide pour l'élimination de ces substances chimiques interférant.
PCT/IN2009/000612 2008-12-15 2009-10-28 Composition pour l'élimination de couleurs et d'inhibiteurs de tissus de plante pour isoler de l'arn WO2010070657A1 (fr)

Applications Claiming Priority (2)

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IN2832DE2008 2008-12-15
IN2832/DEL/2008 2008-12-15

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0554034A1 (fr) * 1992-01-28 1993-08-04 Piotr Chomczynski Produit planche-stable et procédé pour isoler RNA, DNA et proteines
WO2005103252A1 (fr) * 2004-04-16 2005-11-03 Piotr Chomczynski Reactifs et procede d'isolement d'arn purifie
WO2007113614A1 (fr) * 2006-03-30 2007-10-11 Council Of Scientific And Industrial Research Procede d'isolement rapide de l'arn et kit pour ledit isolement

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0554034A1 (fr) * 1992-01-28 1993-08-04 Piotr Chomczynski Produit planche-stable et procédé pour isoler RNA, DNA et proteines
WO2005103252A1 (fr) * 2004-04-16 2005-11-03 Piotr Chomczynski Reactifs et procede d'isolement d'arn purifie
WO2007113614A1 (fr) * 2006-03-30 2007-10-11 Council Of Scientific And Industrial Research Procede d'isolement rapide de l'arn et kit pour ledit isolement

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHRISTOPHER DAY B.: "Genes Involved in Osteoclastogenesis", February 2005, Griffith University Gold Coast Campus School of Medical Sciences, XP002569293 *
JAKOOLA LAURA ET AL: "Isolation of High Quality RNA from Bilberry (Vaccinium myrtillus L.) fruit", MOLECULAR BIOTECHNOLOGY, vol. 19, 2001, pages 201 - 203, XP007911761 *
JONES CHRIS S ET AL: "The isolation of RNA from raspberry (Rubus idaeus) fruit", MOLECULAR BIOTECHNOLOGY, vol. 8, no. 3, December 1997 (1997-12-01), pages 219 - 221, XP007911764, ISSN: 1073-6085 *
LIAO ZHIHUA ET AL: "Rapid isolation of high-quality total RNA from Taxus and Ginkgo", PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, vol. 34, no. 3, 2004, pages 209 - 214, XP007911762, ISSN: 1082-6068 *
TESNIERE C ET AL: "Method For the Isolation of High-Quality RNA from Grape Berry Tissues without Contaminating Tannins or Carbohydrates", PLANT MOLECULAR BIOLOGY REPORTER, vol. 9, no. 3, 1991, pages 242 - 251, XP009129617 *
WOODHEAD MARY ET AL: "Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit", MOLECULAR BIOTECHNOLOGY, vol. 7, no. 1, 1997, pages 1 - 4, XP007911763, ISSN: 1073-6085 *

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