KR102587121B1 - Composition for isolating rna - Google Patents
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Classifications
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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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
Abstract
본 발명은 디메틸설폭사이드 및 구아니딘 티오시아네이트를 포함하는 RNA 분리용 조성물 및 상기 조성물을 이용하여 RNA를 분리하는 방법에 관한 것이다.The present invention relates to a composition for RNA isolation containing dimethyl sulfoxide and guanidine thiocyanate and a method for isolating RNA using the composition.
Description
본 발명은 디메틸설폭사이드 및 구아니딘 티오시아네이트를 포함하는 RNA 분리용 조성물 및 상기 조성물을 이용하여 RNA를 분리하는 방법에 관한 것이다.The present invention relates to a composition for RNA isolation containing dimethyl sulfoxide and guanidine thiocyanate and a method for isolating RNA using the composition.
삶의 질이 향상되면서 질병의 조기 진단에 대한 관심이 커지고 있으며, 분자진단 기술은 질병을 유발하는 병원체의 유전정보(DNA/RNA)를 직접적으로 검출하기 때문에, 기존의 항원/항체 반응을 기반으로 하여 질병의 간접 인자(indirect factor)를 검출하는 면역진단 기술의 단점을 해결할 수 있는 기술로서 많은 관심을 받고 있다. As the quality of life improves, interest in early diagnosis of disease is growing. Molecular diagnostic technology directly detects the genetic information (DNA/RNA) of disease-causing pathogens, based on existing antigen/antibody reactions. Therefore, it is receiving a lot of attention as a technology that can solve the shortcomings of immunodiagnostic technology that detects indirect factors of disease.
또한, 최근 코로나바이러스감염증-19(COVID-19)이 크게 유행하면서 전 세계적으로 많은 사망자가 발생하고 WHO에서는 팬데믹 선언까지 하였다. 이러한 RNA 바이러스에 의한 질병의 경우, 높은 돌연변이 발생률에 의해 더욱 큰 피해가 발생되며 감염 여부에 대한 조기 진단이 더욱 요구되고 있다.In addition, the recent spread of coronavirus infection-19 (COVID-19) has resulted in many deaths around the world, and the WHO even declared it a pandemic. In the case of diseases caused by these RNA viruses, greater damage is caused due to the high mutation rate, and early diagnosis of infection is increasingly required.
한편, miRNA 등 스몰(small) RNA는 생체 내 존재하는 단백질-비 암호화 RNA로, 특정 유전자의 전사 후 과정에 작용하여 해당 유전자의 발현을 조절할 수 있다. 특히, 세포주기, 분화, 발달, 대사, 발암, 노화와 같은 생물학적 기능을 조절하여 생체의 항상성 유지를 매개하는 중요한 유전적 요소로 인지되며, 특히 이의 비정상적인 네트워크 형성은 세포 생리학적인 측면에서 치명적인 결함을 나타낼 수 있다.Meanwhile, small RNAs such as miRNAs are protein-non-coding RNAs that exist in vivo and can regulate the expression of a specific gene by acting on the post-transcriptional process of that gene. In particular, it is recognized as an important genetic element that mediates the maintenance of homeostasis in the living body by regulating biological functions such as cell cycle, differentiation, development, metabolism, carcinogenesis, and aging. In particular, its abnormal network formation causes fatal defects in cell physiology. It can be expressed.
또한, miRNA 등 스몰 RNA의 혈중 내 발현 양상은 암의 초기 단계에서 민감하게 반응하므로 암의 조기, 예측 발견에 있어서 강한 이점을 나타낸다. 또한 단순한 채혈만으로 다양한 암을 검사할 수 있기에 환자로부터 몸에 가해지는 부담이 감소될 수 있다. 나아가, 상기 감염, 암 외에도 알츠하이머, 파킨슨 병 등 여러 난치성 질환의 진단에 있어서 비특이적 검출 반응을 제거하고 RNA를 높은 민감도로 신속하게 검출함으로써 조기 진단이 이루어 질 수 있도록 하는 기술 개발에 대한 요구가 증가되고 있다.In addition, the expression pattern of small RNA such as miRNA in the blood reacts sensitively in the early stages of cancer, providing a strong advantage in early and predictive detection of cancer. Additionally, since a variety of cancers can be tested with a simple blood draw, the burden on the patient's body can be reduced. Furthermore, in the diagnosis of various intractable diseases such as Alzheimer's disease and Parkinson's disease, in addition to the above infections and cancers, there is an increasing demand for the development of technology that enables early diagnosis by eliminating non-specific detection reactions and quickly detecting RNA with high sensitivity. there is.
본 발명의 목적은 RNA 분리용 조성물을 제공하는 것이다.The purpose of the present invention is to provide a composition for RNA isolation.
본 발명의 다른 목적은 상기 RNA 분리용 조성물을 포함하는, RNA 검출용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for detecting RNA, including the composition for separating RNA.
본 발명의 또 다른 목적은 상기 RNA 분리용 조성물을 이용하여 시료로부터 RNA를 분리하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for isolating RNA from a sample using the RNA isolation composition.
상기와 같은 목적을 달성하기 위한 본 발명의 일 측면은, 디메틸설폭사이드(Dimethyl sulfoxide, DMSO); 및 구아니딘 티오시아네이트(Guanidinium thiocyanate, GTC)를 포함하는, RNA 분리용 조성물에 관한 것이다.One aspect of the present invention for achieving the above object is dimethyl sulfoxide (DMSO); and guanidinium thiocyanate (GTC). It relates to a composition for RNA isolation.
본 발명에서, 상기 '분리'는 시료로부터 RNA가 나누어져 떨어지도록 하거나 RNA를 뽑아내는 모든 것을 포함하며, RNA를 '추출'하는 것 역시 시료 내에서 RNA를 분리해내는 것으로서, RNA를 분리하는 것은 '추출'의 상위 개념인 바, 'RNA 추출'은 'RNA 분리'에 포함된다. In the present invention, the 'separation' includes everything that separates the RNA from the sample or extracts the RNA, and 'extracting' the RNA also means separating the RNA from the sample, and isolating the RNA As it is a higher concept of 'extraction', 'RNA extraction' is included in 'RNA isolation'.
구체적으로, 상기 RNA는 스몰(small) RNA 일 수 있다. 상기 스몰 RNA는 miRNA, siRNA 외에도 약 50개 이하의 뉴클레오티드로 이루어진 RNA를 통칭하는 것일 수 있다.Specifically, the RNA may be small RNA. The small RNA may refer to RNA consisting of about 50 nucleotides or less in addition to miRNA and siRNA.
또한 구체적으로, 상기 디메틸설폭사이드는 조성물 총량에 대하여 10 내지 50 %(v/v)로 포함되는 것일 수 있다. Also specifically, the dimethyl sulfoxide may be included in an amount of 10 to 50% (v/v) based on the total amount of the composition.
본 발명 일 실시예에서는 본 발명 조성물 내 포함되는 디메틸설폭사이드의 함량이 증가될수록 동량의 총(total) RNA 추출물 내에 포함되어 있는 small RNA의 양이 증가되는 것을 확인하였으며, 10 내지 50 %(v/v) 전체 범위에서 DMSO가 포함되지 않은 경우에 비해 현저히 증가된 small RNA 함량을 나타내는 것을 확인하였다.In one embodiment of the present invention, it was confirmed that as the content of dimethyl sulfoxide contained in the composition of the present invention increased, the amount of small RNA contained in the same amount of total RNA extract increased, and the amount of small RNA contained in the same amount of total RNA extract increased from 10 to 50% (v/ v) It was confirmed that the small RNA content was significantly increased in the entire range compared to the case where DMSO was not included.
또한 구체적으로, 상기 구아니딘 티오시아네이트는 조성물 총량에 대하여 0.5 내지 4.5 M로 포함되는 것일 수 있다. 더욱 구체적으로 1.0 내지 4.0 M로 포함될 수 있다. Also specifically, the guanidine thiocyanate may be included in an amount of 0.5 to 4.5 M based on the total amount of the composition. More specifically, it may be included from 1.0 to 4.0 M.
본 발명 일 실시예에서는 GTC의 0.5 내지 4.5 M 농도 전범위에서 small RNA가 분리되어 검출되는 것을 확인하였으며, 나아가 1.0 M 내지 4.0 M의 농도 범위에서 small RNA 수득률이 더욱 증가되는 것을 확인하였다.In one embodiment of the present invention, it was confirmed that small RNA was separated and detected in the entire concentration range of 0.5 to 4.5 M of GTC, and further, it was confirmed that the small RNA yield was further increased in the concentration range of 1.0 M to 4.0 M.
또한 구체적으로, 상기 조성물은 아연 이온을 포함하지 않는 것을 특징으로 한다. 본 발명 일 실시예에서는 아연 이온이 포함될 경우 분리되는 small RNA의 양이 아연 이온이 포함되지 않은 경우에 비해 감소되는 것을 확인하였는 바, 본 발명의 RNA 분리용 조성물에는 아연 이온이 포함되지 않는 것을 특징으로 할 수 있다.Also specifically, the composition is characterized in that it does not contain zinc ions. In one embodiment of the present invention, it was confirmed that the amount of small RNA isolated when zinc ions were included was reduced compared to when zinc ions were not included, and the composition for RNA isolation of the present invention does not contain zinc ions. You can do this.
본 발명의 다른 측면은 상기 RNA 분리용 조성물을 포함하는 시료로부터 RNA 검출하기 위한, RNA 검출용 조성물에 관한 것이다.Another aspect of the present invention relates to a composition for detecting RNA from a sample containing the composition for RNA isolation.
본 발명에서, '검출'은 RNA의 존재 유무를 알아내는 것으로서, 본 발명의 RNA 분리용 조성물을 포함하여, RNA를 분리할 수 있도록 함으로써 RNA를 검출할 수 있다.In the present invention, 'detection' refers to determining the presence or absence of RNA, and RNA can be detected by separating RNA, including the composition for RNA isolation of the present invention.
상기 시료는 조직, 세포, 전혈, 혈장, 혈청, 혈액, 타액, 객담, 세포간액 또는 소변과 같은 시료 등을 포함하나 이에 제한되지 않으며, RNA를 포함할 수 있는 시료이면 제한없이 적용될 수 있다. 더욱 구체적으로, 스몰 RNA를 포함하거나, 포함할 것으로 예상되는 시료이면 모두 포함될 수 있다.The sample includes, but is not limited to, samples such as tissue, cells, whole blood, plasma, serum, blood, saliva, sputum, intercellular fluid, or urine, and any sample that can contain RNA can be applied without limitation. More specifically, any sample that contains or is expected to contain small RNA may be included.
본 발명의 또 다른 측면은 a) 디메틸설폭사이드 및 구아니딘 티오시아네이트를 수용액 상태에서 혼합하여 RNA 분리용 조성물을 제조하는 단계; b) 상기 RNA 분리용 조성물을 시료와 혼합하는 단계를 포함하는, 시료로부터 RNA를 분리하는 방법에 관한 것이다.Another aspect of the present invention includes the steps of a) preparing a composition for RNA isolation by mixing dimethyl sulfoxide and guanidine thiocyanate in an aqueous solution; b) It relates to a method of isolating RNA from a sample, comprising the step of mixing the RNA isolation composition with the sample.
상기 시료에서 RNA를 분리하는 과정은 공지의 공정을 이용하여 수행할 수 있으며, 공지의 공정에 본 발명의 조성물을 추가하거나, 본 발명의 조성물만을 단독으로 적용할 수 있다. 본 발명의 조성물이 적용되는 형태는 필요에 따라 변경될 수 있다. 구체적으로, 스몰 RNA를 분리하기 위한 과정에 특징적으로 본 발명의 조성물을 적용할 수 있다.The process of isolating RNA from the sample can be performed using a known process, and the composition of the present invention can be added to the known process, or the composition of the present invention can be applied alone. The form in which the composition of the present invention is applied may be changed as needed. Specifically, the composition of the present invention can be specifically applied to the process for isolating small RNA.
구체적으로, 상기 디메틸설폭사이드는 RNA 분리용 조성물 총량에 대하여 10 내지 50 %(v/v)로 포함되는 것일 수 있으며, 또한 구체적으로 상기 구아니딘 티오시아네이트는 RNA 분리용 조성물 총량에 대하여 0.5 내지 4.5 M로 포함되는 것일 수 있다.Specifically, the dimethyl sulfoxide may be included in an amount of 10 to 50% (v/v) based on the total amount of the RNA isolation composition, and specifically, the guanidine thiocyanate may be included in an amount of 0.5 to 4.5% based on the total amount of the RNA isolation composition. It may be included as M.
RNA 분리 과정에 있어 본 발명의 조성물을 적용함으로써 샘플로부터 수득되는 RNA를 증가시킬 수 있으며, 특히 small RNA의 수득률을 현저히 향상시킬 수 있다. 또한, 본 발명의 조성물을 이용할 경우, 높은 small RNA 분리 효율로 인하여 적은 양의 샘플로도 높은 효율로 small RNA를 검출할 수 있다.By applying the composition of the present invention in the RNA isolation process, the RNA obtained from the sample can be increased, and in particular, the yield of small RNA can be significantly improved. In addition, when using the composition of the present invention, small RNA can be detected with high efficiency even with a small amount of sample due to the high small RNA isolation efficiency.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다. The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 DMSO의 함량에 따른 RNA 검출 변화를 0.8 % 아가로스 젤 전기영동을 통해 확인한 결과를 나타낸 것이다.
도 2는 DMSO의 함량에 따른 small RNA 검출 변화를 15 % 페이지 젤 전기영동을 통해 확인한 결과를 나타낸 것이다(A: 15 % 페이지 젤 전기영동, B: small RNA ratio).
도 3은 GTC의 농도에 따른 RNA 검출 변화를 0.8 % 아가로스 젤 전기영동을 통해 확인한 결과를 나타낸 것이다.
도 4는 GTC의 농도에 따른 small RNA 검출 변화를 15% 페이지 젤 전기영동을 통해 확인한 결과를 나타낸 것이다(A: 15 % 페이지 젤 전기영동, B: small RNA ratio).
도 5는 ZnCl2의 농도에 따른 RNA 검출 변화를 0.8 % 아가로스 젤 전기영동을 통해 확인한 결과를 나타낸 것이다.
도 6은 ZnCl2의 농도에 따른 small RNA 검출 변화를 15 % 페이지 젤 전기영동을 통해 확인한 결과를 나타낸 것이다(A: 15 % 페이지 젤 전기영동, B: small RNA ratio).
도 7은 본 발명 조성물 및 genomic DNA 제거 컬럼을 이용하여 RNA 분리시 genomic DNA가 효과적으로 제거된 것을 확인한 결과를 나타낸 것이다.
도 8은 본 발명 조성물을 이용하여 RNA를 분리한 경우의 small RNA의 함량을 확인한 결과를 나타낸 것이다(A: 15% 페이지 젤 전기영동, B: small RNA ratio).
도 9는 본 발명 조성물을 이용하여 타겟 small RNA를 검출시 검출량 증가 효과를 확인한 결과를 나타낸 것이다(A: hsa-miR-486-5p, B: hsa-miR-21-5p, C: hsa-miR-210).Figure 1 shows the results of confirming changes in RNA detection according to the content of DMSO through 0.8% agarose gel electrophoresis.
Figure 2 shows the results of confirming the change in small RNA detection according to the content of DMSO through 15% Page gel electrophoresis (A: 15% Page gel electrophoresis, B: small RNA ratio).
Figure 3 shows the results of confirming the change in RNA detection according to the concentration of GTC through 0.8% agarose gel electrophoresis.
Figure 4 shows the results of confirming the change in small RNA detection according to the concentration of GTC through 15% Page gel electrophoresis (A: 15% Page gel electrophoresis, B: small RNA ratio).
Figure 5 shows the results of confirming the change in RNA detection according to the concentration of ZnCl 2 through 0.8% agarose gel electrophoresis.
Figure 6 shows the results of confirming the change in small RNA detection according to the concentration of ZnCl 2 through 15% Page gel electrophoresis (A: 15% Page gel electrophoresis, B: small RNA ratio).
Figure 7 shows the results confirming that genomic DNA was effectively removed during RNA isolation using the composition of the present invention and a genomic DNA removal column.
Figure 8 shows the results of confirming the content of small RNA when RNA was isolated using the composition of the present invention (A: 15% Page gel electrophoresis, B: small RNA ratio).
Figure 9 shows the results confirming the effect of increasing the detection amount when detecting target small RNA using the composition of the present invention (A: hsa-miR-486-5p, B: hsa-miR-21-5p, C: hsa-miR -210).
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.
제조예 1. RNA 분리용 조성물의 제조Preparation Example 1. Preparation of composition for RNA isolation
하기와 같이 각 성분을 혼합하여 RNA 분리용 용액(Binding buffer, BD)을 제조하였다. 구체적으로, 용액 총량에 대하여 0 내지 50 %(v/v)의 디메틸설폭사이드(Dimethyl sulfoxide, DMSO) 및 용액 총량에 대하여 0.5 내지 4.5 M의 구아니딘 티오시아네이트(Guanidinium thiocyanate, GTC)를 증류수에서 혼합하여 실시예 1 내지 13의 조성물을 제조하였다. 또한, 용액 총량에 대하여 0 내지 2.0 M의 ZnCl2까지 혼합하여 비교예 1 내지 4의 조성물을 제조하였다.A solution for RNA isolation (Binding buffer, BD) was prepared by mixing each component as follows. Specifically, 0 to 50% (v/v) of dimethyl sulfoxide (DMSO) relative to the total amount of the solution and 0.5 to 4.5 M of guanidinium thiocyanate (GTC) relative to the total amount of the solution are mixed in distilled water. The compositions of Examples 1 to 13 were prepared. In addition, the compositions of Comparative Examples 1 to 4 were prepared by mixing 0 to 2.0 M of ZnCl 2 with respect to the total amount of the solution.
실험예 1. DMSO 함량에 따른 small RNA 분리 효율 확인Experimental Example 1. Confirmation of small RNA isolation efficiency according to DMSO content
상기 제조예를 통해 제조된 본 발명 조성물 내에 DMSO 함량에 따른 small RNA 분리 효율을 확인하였다. The small RNA isolation efficiency according to the DMSO content in the composition of the present invention prepared through the above preparation example was confirmed.
구체적으로, DMSO를 각각 0, 10, 20, 30 및 50 %(v/v)로 포함되도록 하여 상기 실시예 1 내지 5의 조성물을 제조하였으며, 서로 다르게 포함된 DMSO의 함량에 따른 실시예 1 내지 5의 조성물을 적용하여 small RNA를 분리하였다.Specifically, the compositions of Examples 1 to 5 were prepared by containing 0, 10, 20, 30, and 50% (v/v) of DMSO, respectively, and the compositions of Examples 1 to 5 were prepared according to the different amounts of DMSO. Small RNA was isolated by applying the composition of 5.
인간 전혈(whole blood) 샘플로부터 XENOPURE RNA PURIFICATION KIT(cat. No. 93667873-WB)를 이용하여 RNA를 추출하였다. 혈액 200 ul와 XENOSEPA lysis buffer(XENOHELIX, Cat. No. 93667372) 200 ul를 잘 섞은 후 13,500 rpm으로 5 분간 원심분리하였다. 분리된 상층액 200 ul와 클로로포름(chloroform) 100 ul를 잘 섞어준 후 13,500 rpm으로 10 분간 원심분리하였다. 상층액을 genomic DNA 제거용 컬럼 (XENOHELIX, Cat. No. 9366-7622656C4)에 넣고 6,000 rpm으로 1 분간 원심분리하였다.RNA was extracted from human whole blood samples using the XENOPURE RNA PURIFICATION KIT (cat. No. 93667873-WB). 200 ul of blood and 200 ul of XENOSEPA lysis buffer (XENOHELIX, Cat. No. 93667372) were mixed well and centrifuged at 13,500 rpm for 5 minutes. 200 ul of the separated supernatant and 100 ul of chloroform were mixed well and centrifuged at 13,500 rpm for 10 minutes. The supernatant was placed in a column for genomic DNA removal (XENOHELIX, Cat. No. 9366-7622656C4) and centrifuged at 6,000 rpm for 1 minute.
이후 수득된 컬럼 통과액(column flow-through)을 나누어 각각에 상기 실시예 1 내지 5의 RNA 분리용 조성물(BD)을 가하여 혼합한 후 동량의 아이소프로판올(isopropanol)을 혼합하였다. 이 혼합액을 RNA 정제용 컬럼(XENOHELIX, Cat. No. 9366-7622656B4)에 부어준 후 6,000 rpm으로 1 분간 원심분리하였다. 이후 컬럼 통과액은 버리고 워싱버퍼 1(washing buffer 1, XENOHELIX, Cat. No. 9366-2833371) 750 ul을 컬럼에 부어준 후 6,000 rpm으로 1분간 원심분리하였다. 이후 컬럼 통과액은 버리고, 워싱버퍼 2(washing buffer 2, XENOHELIX, Cat. No. 9366-2833372) 750 ul을 컬럼에 부어준 후 6,000 rpm으로 1분간 원심분리하였다. 다시 컬럼 통과액은 버리고, 워싱버퍼 3(washing buffer 3, XENOHELIX, Cat. No. 9366-2833373) 750 ul을 컬럼에 부어준 후 6,000 rpm으로 1분간 원심분리하였다.Afterwards, the obtained column flow-through was divided and the RNA isolation composition (BD) of Examples 1 to 5 was added and mixed to each, followed by mixing an equal amount of isopropanol. This mixture was poured into an RNA purification column (XENOHELIX, Cat. No. 9366-7622656B4) and centrifuged at 6,000 rpm for 1 minute. Afterwards, the column flow liquid was discarded, 750 ul of washing buffer 1 (XENOHELIX, Cat. No. 9366-2833371) was poured into the column, and centrifuged at 6,000 rpm for 1 minute. Afterwards, the column flow liquid was discarded, 750 ul of washing buffer 2 (XENOHELIX, Cat. No. 9366-2833372) was poured into the column, and centrifuged at 6,000 rpm for 1 minute. Again, the column flow liquid was discarded, and 750 ul of washing buffer 3 (XENOHELIX, Cat. No. 9366-2833373) was poured into the column and centrifuged at 6,000 rpm for 1 minute.
RNA 정제용 컬럼 내에서 워싱버퍼를 완전히 제거하기 위하여 컬럼을 새 튜브로 옮긴 후 13,500 rpm으로 3분간 원심분리하였다. 워싱버퍼가 제거된 RNA 정제용 컬럼을 새로운 1.5 ml micro centrifuge tube에 장착한 뒤, 40 ul의 DNase/RNase가 포함되지 않은 증류수를 넣고 상온에서 1 분간 방치 후 13,500 rpm으로 2 분간 원심분리하였다. 분리 정제된 RNA들을 NanoDrop One Microvolume UV-Vis Spectrophotometer(Thermo Scientific)를 사용하여 정량하고 100 ng 씩 0.8% 아가로스 젤(agarose gel)에 전기영동 하였다.To completely remove the washing buffer from the RNA purification column, the column was transferred to a new tube and centrifuged at 13,500 rpm for 3 minutes. The RNA purification column from which the washing buffer was removed was mounted in a new 1.5 ml micro centrifuge tube, then 40 ul of distilled water without DNase/RNase was added, left at room temperature for 1 minute, and then centrifuged at 13,500 rpm for 2 minutes. The separated and purified RNAs were quantified using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific), and 100 ng each was electrophoresed on a 0.8% agarose gel.
그 결과, 도 1에 나타난 바와 같이 조성물 내 포함된 DMSO의 함량이 증가될수록 28s rRNA, 18s rRNA와 같은 큰 사이즈의 RNA의 검출이 감소되고, small RNA의 검출이 증가되는 것을 확인하였다.As a result, as shown in Figure 1, it was confirmed that as the content of DMSO contained in the composition increased, the detection of large-sized RNA such as 28s rRNA and 18s rRNA decreased and the detection of small RNA increased.
또한, 상기 샘플을 15 % 페이지 젤(page gel)에 전기영동하여 분리된 small RNA의 양을 관찰하였다. 그 결과, 도 2에 나타난 바와 같이 조성물 내 포함된 DMSO의 함량이 증가할수록 동량의 총(total) RNA 추출물 내에 포함되어 있는 small RNA의 양이 최대 4.36배까지 증가되는 것을 확인하였다.Additionally, the sample was electrophoresed on a 15% Page gel to observe the amount of separated small RNA. As a result, as shown in Figure 2, it was confirmed that as the content of DMSO contained in the composition increased, the amount of small RNA contained in the same amount of total RNA extract increased up to 4.36 times.
실험예 2. GTC 함량에 따른 small RNA 분리 효율 확인Experimental Example 2. Confirmation of small RNA isolation efficiency according to GTC content
상기 제조예를 통해 제조된 본 발명 조성물 내에 GTC 함량에 따른 small RNA 분리 효율을 확인하였다. The small RNA isolation efficiency according to GTC content was confirmed in the composition of the present invention prepared through the above preparation example.
GTC를 각각 0.5, 1.0, 1.8, 2.5, 3.0, 3.5, 4.0 및 4.5 M로 포함되도록 하여 상기 실시예 6 내지 13의 조성물을 제조하였으며, 서로 다르게 포함된 GTC의 함량에 따른 실시예 6 내지 13의 조성물을 적용하여 small RNA를 분리하였다.The compositions of Examples 6 to 13 were prepared by containing 0.5, 1.0, 1.8, 2.5, 3.0, 3.5, 4.0 and 4.5 M of GTC, respectively, and the compositions of Examples 6 to 13 according to the different amounts of GTC were prepared. The composition was applied to isolate small RNA.
상기 실험예 1과 동일한 방식으로 전혈에서 small RNA를 분리 및 정제하였으며, 실시예 6 내지 13의 조성물을 적용하여 small RNA를 분리하였다.Small RNA was isolated and purified from whole blood in the same manner as in Experimental Example 1, and small RNA was isolated by applying the compositions of Examples 6 to 13.
이후, 분리 정제된 RNA들을 NanoDrop One Microvolume UV-Vis Spectrophotometer(Thermo Scientific)를 사용하여 정량하고 100 ng 씩 0.8 % 아가로스 젤(agarose gel)에 전기영동하였다. 그 결과, 도 3에 나타난 바와 같이 전체 RNA에 있어서는 GTC 농도 증가에 따른 유의미한 수득률 변화는 나타나지 않았다.Afterwards, the separated and purified RNA was quantified using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific) and electrophoresed on a 0.8% agarose gel (100 ng each). As a result, as shown in Figure 3, there was no significant change in yield of total RNA as the GTC concentration increased.
또한, 상기 샘플을 15% 페이지 젤(page gel)에 전기영동하여 분리된 small RNA의 양을 관찰하였다. 그 결과, 도 4에 나타난 바와 같이 GTC의 0.5 내지 4.5 M농도 전범위에서 small RNA가 분리되어 검출되는 것을 확인하였다. 또한, GTC의 농도에 따라 small RNA의 수득률 변화가 나타났으며, 1.0 M 내지 4.0 M의 농도 범위에서 small RNA 수득률이 현저히 증가된 수준으로 나타난 것을 확인하였다.Additionally, the sample was electrophoresed on a 15% Page gel to observe the amount of separated small RNA. As a result, as shown in Figure 4, it was confirmed that small RNA was separated and detected in the entire concentration range of 0.5 to 4.5 M of GTC. In addition, the yield of small RNA changed depending on the concentration of GTC, and it was confirmed that the yield of small RNA was significantly increased in the concentration range of 1.0 M to 4.0 M.
실험예 3. small RNA 분리에서의 아연 이온의 영향 확인Experimental Example 3. Confirmation of the effect of zinc ions on small RNA isolation
상기 제조예를 통해 제조된 본 발명 조성물 내에 아연 이온 포함에 따른 small RNA 분리 효율을 확인하였다.The small RNA isolation efficiency according to the inclusion of zinc ions in the composition of the present invention prepared through the above preparation example was confirmed.
ZnCl2를 각각 0.5, 1.0 및 2.0 M로 포함되도록 하여 비교예 1 내지 3의 조성물을 제조하였으며, 서로 다르게 포함된 ZnCl2의 함량에 따른 상기 비교예 1 내지 3의 조성물을 적용하여 small RNA를 분리하였다.The compositions of Comparative Examples 1 to 3 were prepared by containing 0.5, 1.0, and 2.0 M of ZnCl 2, respectively, and small RNA was isolated by applying the compositions of Comparative Examples 1 to 3 according to the different amounts of ZnCl 2 . did.
상기 실험예 1과 동일한 방식으로 전혈에서 small RNA를 분리 및 정제하였으며, 비교예 1 내지 3의 조성물을 적용하여 small RNA를 분리하였다.Small RNA was isolated and purified from whole blood in the same manner as in Experimental Example 1, and small RNA was isolated by applying the compositions of Comparative Examples 1 to 3.
이후, 분리 정제된 RNA들을 NanoDrop One Microvolume UV-Vis Spectrophotometer(Thermo Scientific)를 사용하여 정량하고 100 ng 씩 0.8 % 아가로스 젤(agarose gel)에 전기영동하였다.Afterwards, the separated and purified RNA was quantified using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific) and electrophoresed on a 0.8% agarose gel (100 ng each).
그 결과, 도 5에 나타난 바와 같이 조성물 내 포함된 아연 이온의 농도가 증가될수록 28s rRNA, 18s rRNA와 같은 큰 사이즈의 RNA의 검출이 감소된 것을 확인하였다.As a result, as shown in Figure 5, it was confirmed that as the concentration of zinc ions contained in the composition increased, the detection of large-sized RNA such as 28s rRNA and 18s rRNA decreased.
또한, 상기 샘플을 15 % 페이지 젤(page gel)에 전기영동하여 분리된 small RNA의 양을 관찰하였다. 그 결과, 도 6에 나타난 바와 같이 조성물 내 아연 이온이 포함된 경우(비교예 1 내지 3), 포함되지 않은 경우(ZnCl2 0 M)에 비해 최대 약 41 % 수준까지 감소되었다. Additionally, the sample was electrophoresed on a 15% Page gel to observe the amount of separated small RNA. As a result, as shown in FIG. 6, when zinc ions were included in the composition (Comparative Examples 1 to 3), the composition was reduced by up to about 41% compared to when it was not included (ZnCl 2 0 M).
상기와 같은 결과는 본 발명의 조성물을 이용하여 RNA를 분리하거나 추출하는 과정에서 아연 이온을 포함하는 경우, RNA 분리 또는 추출이 안정적이기 어려움을 보여준다.The above results show that when zinc ions are included in the process of separating or extracting RNA using the composition of the present invention, it is difficult to separate or extract RNA stably.
실험예 4. 본 발명 조성물의 small RNA 분리 효율 증가 확인Experimental Example 4. Confirmation of increased small RNA isolation efficiency of the composition of the present invention
4-1. 본 발명 조성물(RNA Binding Buffer, BD) 처리에 따른 small RNA 분리 효율 확인4-1. Confirmation of small RNA isolation efficiency according to treatment with the composition of the present invention (RNA Binding Buffer, BD)
인간 전혈(whole blood) 샘플로부터 XENOPURE RNA PURIFICATION KIT(cat. No. 93667873-WB)를 이용하여 RNA를 추출하였다. 혈액 200 ul와 XENOSEPA lysis buffer(XENOHELIX, Cat. No. 93667372) 200 ul를 잘 섞은 후 13,500 rpm으로 5 분간 원심분리하였다. 분리된 상층액 200 ul와 클로로포름(chloroform) 100 ul를 잘 섞어준 후 13,500 rpm으로 10 분간 원심분리하였다. 상층액을 genomic DNA 제거용 컬럼 (XENOHELIX, Cat. No. 9366-7622656C4)에 넣고 6,000 rpm으로 1 분간 원심분리하였다.RNA was extracted from human whole blood samples using the XENOPURE RNA PURIFICATION KIT (cat. No. 93667873-WB). 200 ul of blood and 200 ul of XENOSEPA lysis buffer (XENOHELIX, Cat. No. 93667372) were mixed well and centrifuged at 13,500 rpm for 5 minutes. 200 ul of the separated supernatant and 100 ul of chloroform were mixed well and centrifuged at 13,500 rpm for 10 minutes. The supernatant was placed in a column for genomic DNA removal (XENOHELIX, Cat. No. 9366-7622656C4) and centrifuged at 6,000 rpm for 1 minute.
이후 수득된 컬럼 통과액(column flow-through)을 나누어 각각에 상기 제조예에서 제조한 실시예 5의 RNA 분리용 조성물(BD)을 25, 50, 100 ul을 가하여 혼합한 후, 동량의 아이소프로판올(isopropanol)을 혼합하였다. 이 혼합액을 RNA 정제용 컬럼(XENOHELIX, Cat. No. 9366-7622656B4)에 부어준 후 6,000 rpm으로 1 분간 원심분리하였다. 이후 컬럼 통과액은 버리고 워싱버퍼 1(washing buffer 1, XENOHELIX, Cat. No. 9366-2833371) 750 ul을 컬럼에 부어준 후 6,000 rpm으로 1분간 원심분리하였다. 이후 컬럼 통과액은 버리고, 워싱버퍼 2(washing buffer 2, XENOHELIX, Cat. No. 9366-2833372) 750 ul을 컬럼에 부어준 후 6,000 rpm으로 1분간 원심분리하였다. 다시 컬럼 통과액은 버리고, 워싱버퍼 3(washing buffer 3, XENOHELIX, Cat. No. 9366-2833373) 750 ul을 컬럼에 부어준 후 6,000 rpm으로 1분간 원심분리하였다.Afterwards, the obtained column flow-through was divided and mixed with 25, 50, and 100 ul of the RNA isolation composition (BD) of Example 5 prepared in the above Preparation Example, and then mixed with an equal amount of isopropanol. (isopropanol) was mixed. This mixture was poured into an RNA purification column (XENOHELIX, Cat. No. 9366-7622656B4) and centrifuged at 6,000 rpm for 1 minute. Afterwards, the column flow liquid was discarded, 750 ul of washing buffer 1 (XENOHELIX, Cat. No. 9366-2833371) was poured into the column, and centrifuged at 6,000 rpm for 1 minute. Afterwards, the column flow liquid was discarded, 750 ul of washing buffer 2 (XENOHELIX, Cat. No. 9366-2833372) was poured into the column, and centrifuged at 6,000 rpm for 1 minute. Again, the column flow liquid was discarded, and 750 ul of washing buffer 3 (XENOHELIX, Cat. No. 9366-2833373) was poured into the column and centrifuged at 6,000 rpm for 1 minute.
RNA 정제용 컬럼 내에서 워싱버퍼를 완전히 제거하기 위하여 컬럼을 새 튜브로 옮긴 후 13,500 rpm으로 3분간 원심분리하였다. 워싱버퍼가 제거된 RNA 정제용 컬럼을 새로운 1.5 ml micro centrifuge tube에 장착한 뒤, 40 ul의 Dnase/Rnase가 포함되지 않은 증류수를 넣고 상온에서 1 분간 방치 후 13,500 rpm으로 2 분간 원심분리하였다. 분리 정제된 RNA들을 NanoDrop One Microvolume UV-Vis Spectrophotometer(Thermo Scientific)를 사용하여 정량하고 100 ng 씩 0.8 % 아가로스 젤(agarose gel)에 전기영동 하였다.To completely remove the washing buffer from the RNA purification column, the column was transferred to a new tube and centrifuged at 13,500 rpm for 3 minutes. The RNA purification column from which the washing buffer was removed was mounted in a new 1.5 ml micro centrifuge tube, then 40 ul of distilled water without Dnase/Rnase was added, left at room temperature for 1 minute, and then centrifuged at 13,500 rpm for 2 minutes. The separated and purified RNA was quantified using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific), and 100 ng each was electrophoresed on a 0.8% agarose gel.
그 결과, 도 7에 나타난 바와 같이 genomic DNA 제거 컬럼 및 본 발명의 조성물이 적용된 경우, 분리된 RNA 내에 genomic DNA가 효과적으로 제거되어 분리된 RNA 내에 genomic DNA가 포함되지 않는 것을 확인하였다. As a result, as shown in Figure 7, when the genomic DNA removal column and the composition of the present invention were applied, it was confirmed that genomic DNA was effectively removed from the separated RNA and genomic DNA was not included in the isolated RNA.
또한 상기와 같이 분리한 RNA를 15 % 아가로스 젤에 전기영동하여 small RNA의 함량을 확인하였다. 그 결과, 도 8에 나타난 바와 같이 상기 실시예 5의 조성물을 가하였을 때, 그렇지 않은 경우(BD 0 ul)에 비해 small RNA의 함량이 현저히 증가하는 것을 확인하였으며, 혼합되는 실시예 5의 조성물이 증가할수록 small RNA의 함량 및 비율이 증가되는 것을 확인하였다. 특히 25 ul의 저용량을 혼합하더라도 약 4 배 이상 증가되는 것을 확인하였는 바, 이러한 결과는 본 발명의 조성물이 small RNA 분리 효율을 현저히 증가시킬 수 있음을 나타내는 것이다(도 8A 및 8B).In addition, the RNA isolated as above was electrophoresed on a 15% agarose gel to confirm the content of small RNA. As a result, as shown in Figure 8, it was confirmed that when the composition of Example 5 was added, the content of small RNA was significantly increased compared to the case where it was not added (BD 0 ul), and the composition of Example 5 mixed was confirmed to be It was confirmed that the content and ratio of small RNA increased as it increased. In particular, it was confirmed that the increase was increased by about 4 times even when mixing at a low volume of 25 ul. This result indicates that the composition of the present invention can significantly increase the small RNA isolation efficiency (Figures 8A and 8B).
4-2. 본 발명 조성물 처리에 따른 small RNA의 순도 확인4-2. Confirmation of purity of small RNA according to treatment with the composition of the present invention
상기 분리 정제된 RNA들을 NanoDrop One Spectrophotometer(Thermo Scientific)를 이용하여 정량하였다. The separated and purified RNA was quantified using NanoDrop One Spectrophotometer (Thermo Scientific).
그 결과, 하기 표 4에 나타난 바와 같이 실시예 5의 조성물을 가하였을 때 단백질 및 화합물(chemicals)에 의한 오염 지표에 있어서도 우수한 것으로 나타남에 따라 본 발명의 조성물을 가하였을 때 단백질이나 화합물에 의한 오염 없이 높은 순도로 추출됨을 확인하였다. As a result, as shown in Table 4 below, when the composition of Example 5 was added, it was found to be excellent in terms of contamination indicators by proteins and chemicals, so when the composition of the present invention was added, contamination by proteins or compounds was observed. It was confirmed that it was extracted with high purity.
4-3. 본 발명 조성물 처리에 따른 miRNA 검출량 확인4-3. Confirmation of detected amount of miRNA according to treatment with composition of the present invention
샘플에서 분리되는 small RNA의 분리 효율에 따른 수득량 및 분리된 small RNA의 순도는 miRNA 검출 및 탐지에 큰 영향을 미친다. 이에 본 발명자들은 상기 실시예 5의 조성물을 적용하여 small RNA를 분리하고, 이 중 hsa-miR-486-5p, hsa-miR-21-5p 및 hsa-miR-210가 검출되는 양을 확인하였다. The yield according to the separation efficiency of small RNA isolated from the sample and the purity of the isolated small RNA have a significant impact on detection and detection of miRNA. Accordingly, the present inventors applied the composition of Example 5 to isolate small RNA, and confirmed the amount of hsa-miR-486-5p, hsa-miR-21-5p, and hsa-miR-210 detected.
상기 4-1과 동일한 방식으로 RNA를 추출 및 정제하였다. 이후 XENO-Q miRNA 검출키트(XENOHELIX, Cat. No. 93661000)를 제품 설명서에 따라 이용하여 hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-210의 양을 확인하였다. 실험 방법은 아래와 같다. 1 ug의 전혈 RNA 및 500 amol의 hsa-miR486-5p, hsa-miR21-5p 및 hsa-miR210-3p, 센서 DNA를 혼합하였다. 상기 각 miRNA 검출을 위한 센서 DNA의 서열은 하기 표 5에 정리된 바와 같다.RNA was extracted and purified in the same manner as in 4-1 above. Afterwards, the amounts of hsa-miR-486-5p, hsa-miR-21-5p, and hsa-miR-210 were confirmed using the XENO-Q miRNA detection kit (XENOHELIX, Cat. No. 93661000) according to the product instructions. The experimental method is as follows. 1 ug of whole blood RNA and 500 amol of hsa-miR486-5p, hsa-miR21-5p and hsa-miR210-3p, sensor DNA were mixed. The sequence of the sensor DNA for detecting each of the above-mentioned miRNAs is summarized in Table 5 below.
이후, 2 ㎕의 반응용 완충용액(reaction Buffer, 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, 1% Triton X-100, (pH 8.8 at 25°C)), 1 ㎕의 10 mM dNTP 및 2 unit의 DNA 폴리머라제(XenoT-POL)를 혼합하였다. 혼합액을 95 ℃에서 1분 간 가열한 후 63 ℃ 에서 5 분간 인큐베이션(incubation)하였다. 상기로부터 수득된 샘플 20 ㎕을 MEGAquick-spin??Plus Total Fragment DNA Purification Kit((주)인트론바이오테크놀로지 Cat no. 17290)를 이용하여 클린업하고, 40 ㎕의 증류수로 용리하여, 엔도뉴클레아제 혼합물(40 mM 소듐아세테이트(sodium acetate, pH 4.5 at 25 °C), 300 mM NaCl, 2 mM ZnSO4 및 200 unit S1 뉴클레아제(nuclease))을 섞어주고 30 °C에서 10 분간 인큐베이션 하였다.Afterwards, 2 ㎕ of reaction buffer (200mM Tris-HCl, 100mM ( NH4 ) 2SO4 , 100mMKCl, 20mMMgSO4 , 1% Triton X-100, (pH 8.8 at 25) °C), 1 μl of 10 mM dNTP and 2 units of DNA polymerase (XenoT-POL) were mixed. The mixed solution was heated at 95°C for 1 minute and then incubated at 63°C for 5 minutes. 20 ㎕ of the sample obtained above was cleaned up using the MEGAquick-spin??Plus Total Fragment DNA Purification Kit (Intron Biotechnology Co., Ltd. Cat no. 17290), eluted with 40 ㎕ of distilled water, and endonuclease mixture. (40mM sodium acetate (pH 4.5 at 25°C), 300mM NaCl, 2mM ZnSO 4 and 200 unit S1 nuclease) were mixed and incubated at 30°C for 10 minutes.
이후, MEGAquick-spin??Plus Total Fragment DNA Purification Kit을 이용하여 클린업하고, 200 ㎕의 증류수로 용리한 후 3 ul 샘플을 miR486-5p, miR210-3p, miR21-5p 센서 특이적 프라이머를 이용하여 95 ℃ 3 분 1사이클, 95 ℃ 10초, 64.0 ℃ 30초 40 사이클 조건으로 qPCR을 수행하였다.Afterwards, clean-up was performed using the MEGAquick-spin??Plus Total Fragment DNA Purification Kit, and after elution with 200 ㎕ of distilled water, a 3 ul sample was purified by 95 μl using specific primers for the sensors: qPCR was performed under the following conditions: 1 cycle of 3 minutes at °C, 40 cycles of 95 °C for 10 seconds, and 64.0 °C for 30 seconds.
상기 qPCR 수행시 이용된 프라이머 서열은 하기 표 6에 정리된 바와 같다.Primer sequences used when performing the qPCR are summarized in Table 6 below.
그 결과, 도 9에 나타난 바와 같이, 실시예 5의 조성물을 가하였을 때, 그렇지 않은 경우(BD 0 ul)에 비해 hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR-210 모두 검출되는 양이 증가하였으며, 구체적으로 hsa-miR-486-5p는 약 1.33 배, hsa- miR21-5p는 약 3 배, hsa- miR210은 약 1.87배 증가됨을 확인하였다.As a result, as shown in Figure 9, when the composition of Example 5 was added, hsa-miR-486-5p, hsa-miR-21-5p, hsa-miR- compared to the other case (BD 0 ul) 210 The amount detected was increased for all, and specifically, it was confirmed that hsa-miR-486-5p increased by about 1.33 times, hsa-miR21-5p by about 3 times, and hsa-miR210 by about 1.87 times.
이는 본 발명의 조성물을 이용할 경우, 동일 샘플 내에서 검출될 수 있는small RNA의 양이 증가되는 바, 높은 small RNA 분리 효율로 인하여 기존에 비해 샘플의 양이 적더라도 small RNA의 검출 효율 역시 증가됨을 나타내는 것이다.This means that when using the composition of the present invention, the amount of small RNA that can be detected in the same sample increases, and due to the high small RNA isolation efficiency, the detection efficiency of small RNA also increases even if the amount of sample is smaller than before. It represents.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.
<110> Xenohelix. Co., Ltd. <120> COMPOSITION FOR ISOLATING RNA <130> 20PP31102 <150> KR 10-2020-0118200 <151> 2020-09-15 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA detecting for hsa-miR-486-5p <400> 1 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt taggcgtgac 60 tggagttgct tggctctggt gtattggttc ggggcagctc agtacagga 109 <210> 2 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA detecting for hsa-miR-21-5p <400> 2 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatc gtgatgtgac 60 tggagttgct tggctctggt gtattggttc aacatcagtc tgataagcta 110 <210> 3 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA detecting for hsa-miR-210 <400> 3 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagata catcggtgac 60 tggagttgct tggctctggt gtattggttc agccgctgtc acacgcacag 110 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-486-5p forward primer <400> 4 cgctacagtc gcatacgaga tttaggc 27 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-486-5p reverse primer <400> 5 tcctgtactg agctgccccg ag 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-21-5p forward primer <400> 6 cgcatacgag atcgtgatgt g 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-21-5p reverse primer <400> 7 tagcttatca gactgatgtt gaac 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-210 forward primer <400> 8 ctaagtcgca tacgagatac atcg 24 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-210 reverse primer <400> 9 tgcgtgtgac agcggctgaa c 21 <110> Xenohelix. Co., Ltd. <120> COMPOSITION FOR ISOLATING RNA <130> 20PP31102 <150> KR 10-2020-0118200 <151> 2020-09-15 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA detecting for hsa-miR-486-5p <400> 1 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt taggcgtgac 60 tggagttgct tggctctggt gtattggttc ggggcagctc agtacagga 109 <210> 2 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA detecting for hsa-miR-21-5p <400> 2 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatc gtgatgtgac 60 tggagttgct tggctctggt gtattggttc aacatcagtc tgataagcta 110 <210> 3 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA detecting for hsa-miR-210 <400> 3 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagata catcggtgac 60 tggagttgct tggctctggt gtattggttc agccgctgtc acacgcacag 110 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-486-5p forward primer <400> 4 cgctacagtc gcatacgaga tttaggc 27 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223>hsa-miR-486-5p reverse primer <400> 5 tcctgtactg agctgccccg ag 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hsa-miR-21-5p forward primer <400> 6 cgcatacgag atcgtgatgt g 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223>hsa-miR-21-5p reverse primer <400> 7 tagcttatca gactgatgtt gaac 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223>hsa-miR-210 forward primer <400> 8 ctaagtcgca tacgagatac atcg 24 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223>hsa-miR-210 reverse primer <400> 9 tgcgtgtgac agcggctgaa c 21
Claims (10)
상기 디메틸설폭사이드(Dimethyl sulfoxide, DMSO)는 조성물 총량에 대하여 20 내지 50%(v/v)로 포함되고,
상기 구아니딘 티오시아네이트는 조성물 총량에 대하여 1 내지 4.5M로 포함되는 것인, 스몰(small) RNA 분리용 조성물.Dimethyl sulfoxide (DMSO); A composition for small RNA isolation comprising guanidinium thiocyanate (GTC),
The dimethyl sulfoxide (DMSO) is contained in 20 to 50% (v/v) based on the total amount of the composition,
A composition for small RNA isolation, wherein the guanidine thiocyanate is contained in an amount of 1 to 4.5M based on the total amount of the composition.
상기 조성물은 아연 이온을 포함하지 않는 것을 특징으로 하는 것인, 스몰(small) RNA 분리용 조성물. According to paragraph 1,
A composition for isolating small RNA, characterized in that the composition does not contain zinc ions.
시료로부터 RNA 검출하기 위한, 스몰(small) RNA 검출용 조성물.Comprising the composition of claim 1,
A composition for detecting small RNA, for detecting RNA from a sample.
상기 시료는 조직, 세포, 전혈, 혈장, 혈청, 혈액, 타액, 객담, 세포간액 또는 소변 시료인, 스몰(small) RNA 검출용 조성물.According to clause 6,
The sample is a composition for detecting small RNA, which is a tissue, cell, whole blood, plasma, serum, blood, saliva, sputum, intercellular fluid, or urine sample.
b) 상기 RNA 분리용 조성물을 시료와 혼합하는 단계를 포함하는, 시료로부터 스몰(small) RNA를 분리하는 방법으로서,
상기 디메틸설폭사이드는 조성물 총량에 대하여 20 내지 50%(v/v)로 포함되고,
상기 구아니딘 티오시아네이트는 조성물 총량에 대하여 1 내지 4.5M로 포함되는 것인,
스몰(small) RNA를 분리하는 방법.a) preparing a composition for small RNA isolation by mixing dimethyl sulfoxide and guanidine thiocyanate in an aqueous solution; and
b) A method of isolating small RNA from a sample, comprising the step of mixing the RNA isolation composition with the sample,
The dimethyl sulfoxide is contained in 20 to 50% (v/v) based on the total amount of the composition,
The guanidine thiocyanate is contained in an amount of 1 to 4.5 M based on the total amount of the composition,
Method for isolating small RNA.
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