Title of Invention	A COMPOSITION FOR REMOVAL OF THE COLOURS AND INHIBITORS FROM PLANT TISSUES TO ISOLATE RNA
Abstract	The present invention provides a composition and process for removal of colours and inhibitors from tissues to isolate RNA. Particularly, this invention relates to a composition and the method to remove the soluble substances that otherwise would impart colour to the ribonucleic acid (RNA) preparations or would inhibit down stream applications including synthesis of complementary DNA (cDNA) and polymerase chain reaction using RNA as one of the substrates. Reported protocols till date can isolate RNA, but the isolated RNA is not suitable for downstream processes such as RT-PCR due to presence of colours and the associated interfering agents. None of the protocols describe a simple and quick method for removal of these interfering chemicals

Title of Invention

A COMPOSITION FOR REMOVAL OF THE COLOURS AND INHIBITORS FROM PLANT TISSUES TO ISOLATE RNA

Abstract

The present invention provides a composition and process for removal of colours and inhibitors from tissues to isolate RNA. Particularly, this invention relates to a composition and the method to remove the soluble substances that otherwise would impart colour to the ribonucleic acid (RNA) preparations or would inhibit down stream applications including synthesis of complementary DNA (cDNA) and polymerase chain reaction using RNA as one of the substrates. Reported protocols till date can isolate RNA, but the isolated RNA is not suitable for downstream processes such as RT-PCR due to presence of colours and the associated interfering agents. None of the protocols describe a simple and quick method for removal of these interfering chemicals

Full Text	Field of Invention The present invention relates to a composition for the removal of colours and inhibitors from plant tissues to isolate RNA. Particularly, 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 (hereinafter, referred to as cDNA) and polymerase chain reaction using RNA as one of the substrates. The "soluble substance" in the present invention is in context to the solution used to remove these substances. Background and prior art references to the Invention Isolation of RNA 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, microarrays, RNA mapping, in vitro translation. Northern blot analysis, nuclease protection assays and cDNA library construction. There are several methods described in the literature to isolate RNA from plant tissues [Current Protocols in Molecular Biology by Ausubel, F.M., Brent, R., Kingston, R. E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. Eds,1994. John Wiley and Sons, Inc. USA; Cox, R.A.( 1968) In: Methods in Enzymology, Grosmann, L. and Moldave, K. Eds. Vol.12 B, pp. 120-129, Academic Press, Orlando, FL; R.C. Bugos, V.L. Chiang, X-H. Zhang, E.R. Campbell, O.K. Podila, W.H. Campbell, Biotechniques 19 (1995) 734-737; Chirgwin, J.M., Przybyla, A.E., Macdonald, R.J. and Rutter, W.J. (1979) Biochemistry 18: 5294-5299; Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162:156-159; United states Patent 5,945,515 by Chomczynski, P. (1999); US Patent 5,777,099 by Mehra, M. (1998)]. Recently, Ghawana, S., Singh, K., Riazada, J., Rani, A., Bhardwaj, P. K., and Kumar, S., (Patent filed 0344NF2004/IN Dated 30.03.2006) developed a two-solution system for rapid isolation of RNA composed of solution I [buffer saturated phenol (pH less than 7), SDS (0.1-1%), EDTA (10-20mM), sodium acetate(0.3-0.8M)], and solution II [DEPC(0.001-0.1%) treated deionized water]. Some of the tissues possess very high colour and other interfering chemical substances that often co-precipitate with RNA and make downstream application of RNA problematic. For instance, naphthaquinones (Heide and Tabata, 1987, Photochemistry 26:1645-1650) and anthraquinones (Heide and Leistner, 1983, Phytochemistry 22:659-662) are potent enzyme inactivators. However, none of the methods describe dealing with the tissues containing colours and the associated inhibitors. These molecules co-precipitate along with the RNA and make downstream processing difficult. Purification of RNA using various methods has been described in such cases such as RNeasy technology (RNeasy Plant Mini Kit, Qiagen), NucleoSpin RNA Clean-up (Macherey-Nagel), Norgen's RNA Purification Technology (Norgen Biotek Corp.), Zymo Research's RNA Clean-up Kit (Zymo Research Corp.), RNA purification method using RNACIean (Agencourt Bioscience Corp.) etc. Thus keeping in purview what all has been reported in the prior art, the drawbacks of the prior art may be summarized as: 1. In tissues such as roots of Arnebia euchroma, Daucus carota and Rheum australe, the coloured molecules and other interfering substances precipitate along with RNA preparations. 2. Reported protocols till date can isolate RNA, but the isolated RNA is not suitable for downstream processes such as RT-PCR due to the presence of colours and the associated interfering agents. None of the protocols describe a simple and quick method for removal of these interfering chemicals. Thus, 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. Objects of invention 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. Summary of the Invention Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using TRIzol reagent (Invitrogen) and iRIS system without the composition (coiinmin) and method as claimed in the present invention failed to give good quality of RNA. We assessed the quality of RNA by vahous methods such as absorabance spectrum analysis (using spectrophotometer), eiectrophoresing on denaturating agarose gel and RT-PCR based amplification of 26S rRNA. While with the composition (coiinmin) 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. Accordingly, the present invention provides a composition for the removal of colours and inhibitors from plant tissues comprising: [a] 0.05 to 0.2% of an anionic detergent; [b] 0.25 to 0.56 % of a chelating agent; [c] 70 to 90 % alcohol in diethylpyrocarbonate [DEPC] treated deionized water. 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; [b] adding 2 to 3 ml of the composition as claimed in claim 1 to the powder as obtained in step [a]; [c] grinding the mixture as obtained in step [b] while still frozen so as to make a homogenous mixture; [d] allowing the mixture as obtained in step [c] to thaw with intermittent grinding; [e] centrifuging the thawed mixture as obtained in step [d] at 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant; [f] resuspending the pellet as obtained in step [e] in 1 to 2 ml of the composition as claimed in claim 1 and vortexing; [g] centrifuging the suspension as obtained in step [f] 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant to obtain the desired decolorized tissue. Brief description of figures: Figure 1 represents total RNA isolated from roots of Arnebia euchroma using various combinations as described 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. Figure 2 represents total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using TRIzol reagent (Invitrogen) without (A) and with (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 euchroma, 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 euchroma, Rheum australe and Daucus carota. Figure 5 represents total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using IRIS system without (A) and with (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 euchroma, 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 euchroma, Rheum australe and Daucus carota, IRIS system without (A) and with (B) the claimed composition of the present invention (colinmin) following method as claimed in the present invention Detailed description of invention The present invention provides a composition (named as colinmin) and method for removal of colours and inhibitors from plant tissues to isolate RNA. The 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. 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-lauroyI sarcosine. While 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. In order to prepare 100.0 ml composition, 50-200 mg sodium dodecyl sulphate, 8-15 mM EDTA (pH 8.0) and DEPC (Diethylpyrocarbonate) treated deionized water were mixed together to make 25 ml of solution A. Just pnor to use, 95 to 98% ethanol was mixed with the said solution A in a ratio of 3; 1. Following steps comprised the method to isolate RNA from tisues using the claimed composition: a) Grinding 10-100 mg of plant tissue to a fine powder in liquid nitrogen using a pestle and mortar. b) Adding 2-3 ml of the composition (colinmin) to sample obtained from above step. c) Grinding the mixture while still frozen so as to make a homogenous mixture. d) Allowing It to thaw with intermittent grinding. e) Transferring the sample obtained from above step to two 2 ml microcentrifuge tubes. f) Centrifuging at 13000-14000 rpm for 10-15 minutes at 4-6 degree C. Decanted the supernatant. g) Resuspendirrg the pellet obtained from step (f) in 1-2 ml of composition (colinmin) using a pipette and vortexed for ~ 10 seconds. h) Centrifuging at 13000-14000 rpm for 5-10 minutes at 4-6 degree C. Decanted the supernatant, i) Pellet thus obtained from step (h) can be processed further for RNA isolation following protocol such as iRIS system and TRIzol reagent (Invitrogen). In an embodiment of the present invention, the said tissue is preferably the roots of Arnebia euchroma, Rheum australe, and Daucus carota. In a further embodiment, the anionic detergent is selected from the group consisting of dodecyl sulphate salt or N-lauroyI sarcosine. In another embodiment of the present invention, the anionic detergent is preferably sodium dodecyl sulphate. In another embodiment of the present invention, the chelating agent is selected from the group consisting of disodium and dipotassium of ethylene diamine tetra acetic acid. In another embodiment of the present invention, the chelating agent is preferably EDTA. In another embodiment of the present invention, the concentration of diethylpyrocarbonate used is about 0.1% volume /volume. In a further embodiment 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. Tablel: RNA yield (µq/100mq tissue) using IRIS System and TRIzol Reagent (Invitrogen) with and without the composition of the present invention (Table Removed) EXAMPLES The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention. EXAMPLE 1 - CowiPsositton of present invention 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. To exemplify, for the preparation of 100 ml composition: 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. Protocol claimed in the present patent followed with the different solution (varying ranges of components claimed along with some other components as given in table 2). Pellet obtained from step (8) was further processed for RNA isolation using iRIS system. Table 2. Comparative analysis of various combinations of solutions tested for removal of colours and inhibitors to isolate RNA. (Table Removed) Total RNA isolated from roots of Arnebia euchroma using solution l-XI (given in table 2) as visible on the gel (Figure 1A). RNA quality and yield was found to be good with solution II, III, VII and VIII. RT-PCR based amplification of 26S rRNA (Figure 1B) 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. EXAMPLE 2 - Protocol for RNA isolation using the composition of the present invention 1. Ground 10-100 mg of plant tissue to a fine powder in liquid nitrogen using a pestle and mortar. 2. Add 2-3 ml of the composition (colinmin) as described under Example 1. 3. Ground the mixture while still frozen so as to make a homogenous mixture. 4. Allow it to thaw with intermittent gnnding, 5. Transfer the sample obtained from above step to two 2 ml microcentrifuge tubes. 6. Centrifuge at 13000-14000 rpm for 10-15 minutes at 4-6 degree C. Decanted the supernatant 7. Resuspend the pellet obtained from step (6) in 1-2 ml of the composition (colinmin) using a pipette and vortexed for ~ 10 seconds. 8. Centrifuge at 13000-14000 rpm for 5-10 minutes at 4-6 degree C. Decant the supernatant. 9. Pellet thus obtained from step (8) is further processed for RNA isolation. 10. We compared the efficacy of two kits for the purpose; TRIzol reagent (Invitrogen) and the IRIS system (Ghawana et al., 2004). 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. Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using the composition (colinmin) and method as claimed in the present invention gave good quality of RNA without co-precipitation of colours and soluble inhibitors. EXAMPLE 3 - Various RNA isolation protocols were followed for companson of efficacy Protocol for RNA isolation using TRIzol Reagent (Invitrogen); 1. Plant Tissue (100 mg) was ground in liquid nitrogen to fine powder. 2. Added 1 ml of TRIzol reagent and further ground to a fine powder using pestle and mortar. 3. Incubated the homogenized samples for 5 min at 15 to 30°C. 4. Added 0.2 ml of chloroform per ml of TRIzol reagent. The tubes were shaken vigorously for 15 seconds and incubated at 15 to 30°C for 2 to 3 min. 5. Centrifuged the samples at 12,000 X g for 15 min at 4°C 6. Transferred the aqueous phase to a fresh tube. 7. Added 0.5 ml of isopropyl alcohol per 1 ml of TRIzol reagent used for initial homogenization. 8. Incubated the samples at 15 to 30°C for 10 min. 9. Centrifuged the samples at 12,000 X g for 10 min at 4°C. 10. Removed the supernatant. Washed the RNA pellet with 75% ethanol and air dry. 11. Dissolved RNA in 30-50 µl of RNase-free water. Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using TRIzol reagent (Figure 2) with the composition (colinmin) and method as claimed in the present invention, the remarkable improvement in the quality of RNA was observed with no co-precipitation of colour and inhibitory substances, lower absorption spectrum in visual range (Figure 3) and intact bands of RNA on denaturating agarose gel. Protocol for RNA isolation using iRIS system: a. plant tissue (100 mg) was ground in liquid nitrogen using mortar and pestle to make fine powder. b. Added 2 ml of solution I. c. Homogenized to make a fine powder in pestle. d. Added 800 pi solution II. e. Transferred sample into two 2.0 ml eppendorf tubes. f. Added 200 pi of chloroform, g. Vortexed and left at room temperature for 10 minutes, h. Centrifuged for 10 minutes at room temperature. i. Transferred upper aqueous phase into a new 2.0 ml eppendorf tube. j. Added 0.6 Volumes of isopropanol. k. Vortexed and left at room temperature for 10 minutes. I. Centrifuged for five minutes at 4°C. m. Resulting RNA pellet was washed with 70% ethanol, air-dried and dissolved in appropriate amount of DEPC treated water. Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using iRIS system (Figure 5) with the composition (colinmin) and method as claimed in the present invention were of high quality similar to the result obtained with TRIzol as evident by absorption spectrum (Figure 6) and RT-PCR (Figure 7). 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. To check the integrity of RNA, 5-6 pg of RNA mixed with 10 pi 2x RNA Loading dye (GenHunter, U.S.A.) and incubated at 65 degree 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 descnbed 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. EXAMPLE 4 - In case of plant tissues containing colours and inhibitors e.g. roots of Arnebia euchroma, Rheum australe and Daucus carota, total RNA isolated using TRIzol reagent (Invitrogen) and iRIS system with the composition (colinmin) and method claimed in the present invention as described in Example 1-2 and Table 1 describes the yields obtained from different tissues. EXAMPLE 5 Reverse transcription-polymerase chain reaction (RT-PCR) was performed to assess the quality of RNA for downstream applications using the RNA from roots of Arnebia euchroma, Daucus carota and Rheum australe. Total RNA (2 pg) was digested with DNase I (Cat. No. 19303007 Promega, U.S.A.) and reverse-transcribed with AccuScript High Fidelity reverse transcriptase (Cat. No.600089-54, Stratagene, U.S.A.) to synthesize cDNA using oligo-dT primer (Cat. No.200820-52, Stratagene, U.S.A.).The manufacturer's instructions were followed in both the above reactions. For PCR amplification, oligonucleotide primers specific to 26S rRNA (an internal control) 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 µl 10X PCR buffer (20 mM Tris-HCI, pH 8.4, 50 Mm KCL, 1.5 mM MgCl2), 0.5 µl Deoxynucleotide triphosphate (dNTPs,10 mM), 1.0 µl Forward primer (10 µM), 1.0µI Reverse primer(10 µM), 1.0 µl cDNA, 0.25 µl Taq DNA polymerase (5U/µL) and 18.75 pi sterile distilled water. The RT-PCR based amplification of 26S rRNA obtained in case of total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using the composition (coltnmin) and method as claimed in the present invention, while the RT-PCR failed to give good amplification with total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota without the composition (colinmin) and method as claimed in the present invention (Figure 4 and 7). The main advantages of the present invention are: 1. Present invention ensures removal of colours and inhibitors from plant tissues. 2. 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. 4. Present invention is less hazardous, cost effective and time saving. We Claim: 1. A composition for the removal of colours and inhibitors from plant tissues to isolate RNA, wherein the said composition comphsing: [a] 0.05 to 0.2% of an anionic detergent; [b] 0.25 to 0.56 % of a chelating agent; [c] 70 to 90 % alcohol in diethylpyrocarbonate [DEPC] treated deionized water. 2. A composition as claimed in claim 1, wherein the anionic detergent is selected from the group consisting of dodecyl sulphate salt or N-lauroyI sarcosine, 3. A composition as claimed in claim 1, wherein the anionic detergent is preferably sodium dodecyl sulphate. 4. A composition as claimed in claim 1, wherein the chelating agent is selected from the group consisting of disodium and dipotassium of ethylene diamine tetra acetic acid. 5. A composition as claimed in claim 1, wherein the chelating agent is preferably EDTA. 6. A composrtion as claimed in claim 1, wherein the concentration of diethylpyrocarbonate used is 0.1% volume /volume. 7. 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; [b] adding 2 to 3 ml of the composition as claimed in claiml to the powder as obtained in step [a]; [c] grinding the mixture as obtained in step [b] while still frozen so as to make a homogenous mixture; [d] allowing the mixture as obtained in step [c] to thaw with intermittent grinding; [e] centrifuging the thawed mixture as obtained in step [d] at 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant; [f] resuspending the pellet as obtained in step [e] in 1 to 2 ml of the composition as claimed in claiml and vortexing; [g] centrifuging the suspension as obtained in step [f\| 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant to obtain the desired decolorized tissue 8. A process as claimed in claim 7, wherein the tissue is preferably selected from the roots of Rheum australe, Arnebia euchroma and Daucus carota. 9. A kit for the removal of colours and inhibitors from plant tissues to isolate RNA, wherein the said kit comprising: a) a composition as claimed in claim 1, and b) instructicms for using the kit. 10. A composition and process for the removal of colours and inhibitors from plant tissues to isolate RNA substantially as herein described with reference to the foregoing examples.

Full Text

Field of Invention
The present invention relates to a composition for the removal of colours and inhibitors from plant tissues to isolate RNA. Particularly, 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 (hereinafter, referred to as cDNA) and polymerase chain reaction using RNA as one of the substrates. The "soluble substance" in the present invention is in context to the solution used to remove these substances.
Background and prior art references to the Invention
Isolation of RNA 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, microarrays, RNA mapping, in vitro translation. Northern blot analysis, nuclease protection assays and cDNA library construction.
There are several methods described in the literature to isolate RNA from plant tissues [Current Protocols in Molecular Biology by Ausubel, F.M., Brent, R., Kingston, R. E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. Eds,1994. John Wiley and Sons, Inc. USA; Cox, R.A.( 1968) In: Methods in Enzymology, Grosmann, L. and Moldave, K. Eds. Vol.12 B, pp. 120-129, Academic Press, Orlando, FL; R.C. Bugos, V.L. Chiang, X-H. Zhang, E.R. Campbell, O.K. Podila, W.H. Campbell, Biotechniques 19 (1995) 734-737; Chirgwin, J.M., Przybyla, A.E., Macdonald, R.J. and Rutter, W.J. (1979) Biochemistry 18: 5294-5299; Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162:156-159; United

states Patent 5,945,515 by Chomczynski, P. (1999); US Patent 5,777,099 by Mehra, M. (1998)]. Recently, Ghawana, S., Singh, K., Riazada, J., Rani, A., Bhardwaj, P. K., and Kumar, S., (Patent filed 0344NF2004/IN Dated 30.03.2006) developed a two-solution system for rapid isolation of RNA composed of solution I [buffer saturated phenol (pH less than 7), SDS (0.1-1%), EDTA (10-20mM), sodium acetate(0.3-0.8M)], and solution II [DEPC(0.001-0.1%) treated deionized water].
Some of the tissues possess very high colour and other interfering chemical substances that often co-precipitate with RNA and make downstream application of RNA problematic. For instance, naphthaquinones (Heide and Tabata, 1987, Photochemistry 26:1645-1650) and anthraquinones (Heide and Leistner, 1983, Phytochemistry 22:659-662) are potent enzyme inactivators. However, none of the methods describe dealing with the tissues containing colours and the associated inhibitors. These molecules co-precipitate along with the RNA and make downstream processing difficult. Purification of RNA using various methods has been described in such cases such as RNeasy technology (RNeasy Plant Mini Kit, Qiagen), NucleoSpin RNA Clean-up (Macherey-Nagel), Norgen's RNA Purification Technology (Norgen Biotek Corp.), Zymo Research's RNA Clean-up Kit (Zymo Research Corp.), RNA purification method using RNACIean (Agencourt Bioscience Corp.) etc.
Thus keeping in purview what all has been reported in the prior art, the drawbacks of the prior art may be summarized as:

1. In tissues such as roots of Arnebia euchroma, Daucus carota and Rheum australe, the coloured molecules and other interfering substances precipitate along with RNA preparations.
2. Reported protocols till date can isolate RNA, but the isolated RNA is not suitable for downstream processes such as RT-PCR due to the presence of colours and the associated interfering agents. None of the protocols describe a simple and quick method for removal of these interfering chemicals.
Thus, 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.
Objects of invention
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.

Summary of the Invention
Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using TRIzol reagent (Invitrogen) and iRIS system without the composition (coiinmin) and method as claimed in the present invention failed to give good quality of RNA. We assessed the quality of RNA by vahous methods such as absorabance spectrum analysis (using spectrophotometer), eiectrophoresing on denaturating agarose gel and RT-PCR based amplification of 26S rRNA. While with the composition (coiinmin) 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.
Accordingly, the present invention provides a composition for the removal of colours and inhibitors from plant tissues comprising:
[a] 0.05 to 0.2% of an anionic detergent;
[b] 0.25 to 0.56 % of a chelating agent;
[c] 70 to 90 % alcohol in diethylpyrocarbonate [DEPC] treated deionized water.
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;

[b] adding 2 to 3 ml of the composition as claimed in claim 1 to the powder as obtained in step [a];
[c] grinding the mixture as obtained in step [b] while still frozen so as to make a homogenous mixture;
[d] allowing the mixture as obtained in step [c] to thaw with intermittent grinding;
[e] centrifuging the thawed mixture as obtained in step [d] at 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant;
[f] resuspending the pellet as obtained in step [e] in 1 to 2 ml of the composition as claimed in claim 1 and vortexing;
[g] centrifuging the suspension as obtained in step [f] 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant to obtain the desired decolorized tissue.
Brief description of figures:
Figure 1 represents total RNA isolated from roots of Arnebia euchroma using various combinations as described 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.
Figure 2 represents total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using TRIzol reagent (Invitrogen) without (A) and with (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 euchroma, 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 euchroma, Rheum australe and Daucus carota.
Figure 5 represents total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using IRIS system without (A) and with (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 euchroma, 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 euchroma, Rheum australe and Daucus carota, IRIS system without (A) and with (B) the claimed composition of the present invention (colinmin) following method as claimed in the present invention
Detailed description of invention
The present invention provides a composition (named as colinmin) and method for removal of colours and inhibitors from plant tissues to isolate RNA.

The 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. 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-lauroyI sarcosine. While 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.
In order to prepare 100.0 ml composition, 50-200 mg sodium dodecyl sulphate, 8-15 mM EDTA (pH 8.0) and DEPC (Diethylpyrocarbonate) treated deionized water were mixed together to make 25 ml of solution A. Just pnor to use, 95 to 98% ethanol was mixed with the said solution A in a ratio of 3; 1.
Following steps comprised the method to isolate RNA from tisues using the claimed composition:
a) Grinding 10-100 mg of plant tissue to a fine powder in liquid nitrogen using a pestle and mortar.
b) Adding 2-3 ml of the composition (colinmin) to sample obtained from above step.
c) Grinding the mixture while still frozen so as to make a homogenous mixture.
d) Allowing It to thaw with intermittent grinding.
e) Transferring the sample obtained from above step to two 2 ml microcentrifuge tubes.

f) Centrifuging at 13000-14000 rpm for 10-15 minutes at 4-6 degree C. Decanted the supernatant.
g) Resuspendirrg the pellet obtained from step (f) in 1-2 ml of composition (colinmin) using a pipette and vortexed for ~ 10 seconds.
h) Centrifuging at 13000-14000 rpm for 5-10 minutes at 4-6 degree C.
Decanted the supernatant, i) Pellet thus obtained from step (h) can be processed further for RNA
isolation following protocol such as iRIS system and TRIzol reagent
(Invitrogen).
In an embodiment of the present invention, the said tissue is preferably the roots of
Arnebia euchroma, Rheum australe, and Daucus carota.
In a further embodiment, the anionic detergent is selected from the group
consisting of dodecyl sulphate salt or N-lauroyI sarcosine.
In another embodiment of the present invention, the anionic detergent is preferably
sodium dodecyl sulphate.
In another embodiment of the present invention, the chelating agent is selected
from the group consisting of disodium and dipotassium of ethylene diamine tetra
acetic acid.
In another embodiment of the present invention, the chelating agent is preferably
EDTA.
In another embodiment of the present invention, the concentration of
diethylpyrocarbonate used is about 0.1% volume /volume.
In a further embodiment 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.
Tablel: RNA yield (µq/100mq tissue) using IRIS System and TRIzol Reagent (Invitrogen) with and without the composition of the present invention
(Table Removed)

EXAMPLES
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention.
EXAMPLE 1 - CowiPsositton of present invention
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. To exemplify, for the preparation of 100 ml composition: 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.

Protocol claimed in the present patent followed with the different solution (varying ranges of components claimed along with some other components as given in table 2). Pellet obtained from step (8) was further processed for RNA isolation using iRIS system.
Table 2. Comparative analysis of various combinations of solutions tested for removal of colours and inhibitors to isolate RNA.
(Table Removed)
Total RNA isolated from roots of Arnebia euchroma using solution l-XI (given in table 2) as visible on the gel (Figure 1A). RNA quality and yield was found to be

good with solution II, III, VII and VIII. RT-PCR based amplification of 26S rRNA (Figure 1B) 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.
EXAMPLE 2 - Protocol for RNA isolation using the composition of the present invention
1. Ground 10-100 mg of plant tissue to a fine powder in liquid nitrogen using a pestle and mortar.
2. Add 2-3 ml of the composition (colinmin) as described under Example 1.
3. Ground the mixture while still frozen so as to make a homogenous mixture.
4. Allow it to thaw with intermittent gnnding,
5. Transfer the sample obtained from above step to two 2 ml microcentrifuge tubes.
6. Centrifuge at 13000-14000 rpm for 10-15 minutes at 4-6 degree C. Decanted the supernatant
7. Resuspend the pellet obtained from step (6) in 1-2 ml of the composition (colinmin) using a pipette and vortexed for ~ 10 seconds.
8. Centrifuge at 13000-14000 rpm for 5-10 minutes at 4-6 degree C. Decant the supernatant.
9. Pellet thus obtained from step (8) is further processed for RNA isolation.
10. We compared the efficacy of two kits for the purpose; TRIzol reagent (Invitrogen) and the IRIS system (Ghawana et al., 2004).

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. Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using the composition (colinmin) and method as claimed in the present invention gave good quality of RNA without co-precipitation of colours and soluble inhibitors.
EXAMPLE 3 - Various RNA isolation protocols were followed for companson of efficacy
Protocol for RNA isolation using TRIzol Reagent (Invitrogen);
1. Plant Tissue (100 mg) was ground in liquid nitrogen to fine powder.
2. Added 1 ml of TRIzol reagent and further ground to a fine powder using pestle and mortar.
3. Incubated the homogenized samples for 5 min at 15 to 30°C.
4. Added 0.2 ml of chloroform per ml of TRIzol reagent. The tubes were shaken vigorously for 15 seconds and incubated at 15 to 30°C for 2 to 3 min.
5. Centrifuged the samples at 12,000 X g for 15 min at 4°C
6. Transferred the aqueous phase to a fresh tube.
7. Added 0.5 ml of isopropyl alcohol per 1 ml of TRIzol reagent used for initial homogenization.
8. Incubated the samples at 15 to 30°C for 10 min.
9. Centrifuged the samples at 12,000 X g for 10 min at 4°C.

10. Removed the supernatant. Washed the RNA pellet with 75% ethanol and air dry.
11. Dissolved RNA in 30-50 µl of RNase-free water.
Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using TRIzol reagent (Figure 2) with the composition (colinmin) and method as claimed in the present invention, the remarkable improvement in the quality of RNA was observed with no co-precipitation of colour and inhibitory substances, lower absorption spectrum in visual range (Figure 3) and intact bands of RNA on denaturating agarose gel.
Protocol for RNA isolation using iRIS system:
a. plant tissue (100 mg) was ground in liquid nitrogen using mortar and pestle
to make fine powder.
b. Added 2 ml of solution I.
c. Homogenized to make a fine powder in pestle.
d. Added 800 pi solution II.
e. Transferred sample into two 2.0 ml eppendorf tubes.
f. Added 200 pi of chloroform,
g. Vortexed and left at room temperature for 10 minutes,
h. Centrifuged for 10 minutes at room temperature.
i. Transferred upper aqueous phase into a new 2.0 ml eppendorf tube.
j. Added 0.6 Volumes of isopropanol.
k. Vortexed and left at room temperature for 10 minutes.
I. Centrifuged for five minutes at 4°C.

m. Resulting RNA pellet was washed with 70% ethanol, air-dried and dissolved in appropriate amount of DEPC treated water.
Total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using iRIS system (Figure 5) with the composition (colinmin) and method as claimed in the present invention were of high quality similar to the result obtained with TRIzol as evident by absorption spectrum (Figure 6) and RT-PCR (Figure 7).
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. To check the integrity of RNA, 5-6 pg of RNA mixed with 10 pi 2x RNA Loading dye (GenHunter, U.S.A.) and incubated at 65 degree 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 descnbed 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.
EXAMPLE 4 -
In case of plant tissues containing colours and inhibitors e.g. roots of Arnebia euchroma, Rheum australe and Daucus carota, total RNA isolated using TRIzol reagent (Invitrogen) and iRIS system with the composition (colinmin) and method

claimed in the present invention as described in Example 1-2 and Table 1 describes the yields obtained from different tissues.
EXAMPLE 5
Reverse transcription-polymerase chain reaction (RT-PCR) was performed to assess the quality of RNA for downstream applications using the RNA from roots of Arnebia euchroma, Daucus carota and Rheum australe. Total RNA (2 pg) was digested with DNase I (Cat. No. 19303007 Promega, U.S.A.) and reverse-transcribed with AccuScript High Fidelity reverse transcriptase (Cat. No.600089-54, Stratagene, U.S.A.) to synthesize cDNA using oligo-dT primer (Cat. No.200820-52, Stratagene, U.S.A.).The manufacturer's instructions were followed in both the above reactions. For PCR amplification, oligonucleotide primers specific to 26S rRNA (an internal control) 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 µl 10X PCR buffer (20 mM Tris-HCI, pH 8.4, 50 Mm KCL, 1.5 mM MgCl2), 0.5 µl Deoxynucleotide triphosphate (dNTPs,10 mM), 1.0 µl Forward primer (10 µM), 1.0µI Reverse primer(10 µM), 1.0 µl cDNA, 0.25 µl Taq DNA polymerase (5U/µL) and 18.75 pi sterile distilled water. The RT-PCR based amplification of 26S rRNA obtained in case of total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota using the composition (coltnmin) and method as claimed in the present invention, while the RT-PCR failed to give good amplification with total RNA isolated from roots of Arnebia euchroma, Rheum australe and Daucus carota without the composition (colinmin) and method as claimed in the present invention (Figure 4 and 7).

The main advantages of the present invention are:
1. Present invention ensures removal of colours and inhibitors from plant tissues.
2. 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.
4. Present invention is less hazardous, cost effective and time saving.

We Claim:
1. A composition for the removal of colours and inhibitors from plant tissues to
isolate RNA, wherein the said composition comphsing:
[a] 0.05 to 0.2% of an anionic detergent;
[b] 0.25 to 0.56 % of a chelating agent;
[c] 70 to 90 % alcohol in diethylpyrocarbonate [DEPC] treated deionized water.

2. A composition as claimed in claim 1, wherein the anionic detergent is selected from the group consisting of dodecyl sulphate salt or N-lauroyI sarcosine,
3. A composition as claimed in claim 1, wherein the anionic detergent is preferably sodium dodecyl sulphate.
4. A composition as claimed in claim 1, wherein the chelating agent is selected from the group consisting of disodium and dipotassium of ethylene diamine tetra acetic acid.
5. A composition as claimed in claim 1, wherein the chelating agent is preferably EDTA.
6. A composrtion as claimed in claim 1, wherein the concentration of diethylpyrocarbonate used is 0.1% volume /volume.
7. 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;
[b] adding 2 to 3 ml of the composition as claimed in claiml to the powder as obtained in step [a];

[c] grinding the mixture as obtained in step [b] while still frozen so as to make a homogenous mixture;
[d] allowing the mixture as obtained in step [c] to thaw with intermittent grinding;
[e] centrifuging the thawed mixture as obtained in step [d] at 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant;
[f] resuspending the pellet as obtained in step [e] in 1 to 2 ml of the composition as claimed in claiml and vortexing;
[g] centrifuging the suspension as obtained in step [f| 13000 to 14000 rpm for 5 to 10 min at a temperature of 4 to 6 degree C and decanting the supernatant to obtain the desired decolorized tissue

8. A process as claimed in claim 7, wherein the tissue is preferably selected from the roots of Rheum australe, Arnebia euchroma and Daucus carota.
9. A kit for the removal of colours and inhibitors from plant tissues to isolate RNA, wherein the said kit comprising:

a) a composition as claimed in claim 1, and
b) instructicms for using the kit.
10. A composition and process for the removal of colours and inhibitors from
plant tissues to isolate RNA substantially as herein described with reference
to the foregoing examples.

Documents:

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Patent Number

279365

Indian Patent Application Number

2832/DEL/2008

PG Journal Number

03/2017

Publication Date

20-Jan-2017

Grant Date

19-Jan-2017

Date of Filing

15-Dec-2008

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	SANJAY KUMAR	INSTITUTE OF HIMALAYAN BIORESOURCE TECHNOLOGY, POST BOX NO.-6, PALAMPUR-176 061 (HP)
2	RAVI SHANKAR SINGH	INSTITUTE OF HIMALAYAN BIORESOURCE TECHNOLOGY, POST BOX NO.-6, PALAMPUR-176 061 (HP)

PCT International Classification Number

C12Q1/68; C12P19/34

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA