KR20220106525A - A method of detecting target rna comprising silica treatment - Google Patents
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Abstract
Description
본 발명은 실리카 처리를 포함하는 표적 RNA 검출 방법, 타겟 RNA의 상보적인 서열을 포함하는 센서 DNA; 및 실리카를 포함하는, RNA 검출용 조성물. 및 실리카 처리를 포함하는 단일가닥 DNA 제거방법에 관한 것이다. The present invention relates to a target RNA detection method comprising a silica treatment, a sensor DNA comprising a complementary sequence of the target RNA; And a composition for detecting RNA, comprising silica. And it relates to a single-stranded DNA removal method comprising a silica treatment.
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 diseases is growing, and molecular diagnosis technology directly detects the genetic information (DNA/RNA) of pathogens that cause disease. Therefore, it is receiving a lot of attention as a technology that can solve the shortcomings of immunodiagnostic technology that detects indirect factors of diseases.
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 coronavirus infection-19 (COVID-19) has caused a lot of deaths worldwide, and the WHO even declared a pandemic. In the case of diseases caused by these RNA viruses, greater damage is caused by a high mutation rate, and early diagnosis of infection is further 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.On the other hand, small (small) RNA such as miRNA is a protein-non-coding RNA that exists in a living body, and can control the expression of a specific gene by acting on the post-transcriptional process of a specific gene. In particular, it is recognized as an important genetic element mediating the maintenance of homeostasis in the living body by regulating biological functions such as cell cycle, differentiation, development, metabolism, carcinogenesis, and aging. can indicate
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 제거하고RNA를 높은 민감도로 신속하게 검출함으로써 조기 진단이 이루어 질 수 있도록 하는 기술 개발에 대한 요구가 증가되고 있다.In addition, the expression pattern of small RNAs such as miRNAs in blood reacts sensitively in the early stages of cancer, thus showing a strong advantage in early and predictive detection of cancer. In addition, the burden on the body from the patient can be reduced because various cancers can be tested with only a simple blood collection. Furthermore, in the diagnosis of various intractable diseases such as Alzheimer's and Parkinson's disease, in addition to the infection and cancer, there is an increasing demand for technology development that enables early diagnosis by eliminating non-specific detection reactions and rapidly detecting RNA with high sensitivity. have.
본 발명의 목적은 실리카를 처리하는 단계를 포함하는, RNA 검출 방법을 제공하는 것이다.It is an object of the present invention to provide a method for detecting RNA, comprising the step of treating silica.
본 발명의 다른 목적은 타겟 RNA의 상보적인 서열을 포함하는 센서 DNA; 및 실리카를 포함하는, RNA 검출용 조성물을 제공하는 것이다.Another object of the present invention is a sensor DNA comprising a complementary sequence of a target RNA; And to provide a composition for detecting RNA, comprising silica.
본 발명의 또 다른 목적은 실리카를 처리하는 단계를 포함하는, 단일가닥 DNA(single strand DNA, ssDNA) 제거 방법을 제공하는 것이다.Another object of the present invention is to provide a method for removing single-stranded DNA (ssDNA), comprising the step of treating silica.
상기와 같은 목적을 달성하기 위한 본 발명의 일 측면은, a) 검출 대상이 되는 타겟 RNA의 상보적인 서열을 포함하는 센서DNA를 타겟 RNA와 혼성화하는 단계; b) 상기 센서 DNA의 모듈 영역을 주형으로 하고, 상기 타겟 RNA를 프라이머로 하여 중합효소로 중합하는 단계; 및 c) 실리카를 처리하여 타겟 RNA와 혼성화되지 않은 센서 DNA를 제거하는 단계를 포함하는, RNA 검출 방법에 관한 것이다.One aspect of the present invention for achieving the above object is, a) hybridizing the sensor DNA comprising the complementary sequence of the target RNA to be detected with the target RNA; b) using the module region of the sensor DNA as a template and polymerizing the target RNA as a primer with a polymerase; and c) treating the silica to remove the sensor DNA that is not hybridized with the target RNA.
구체적으로, 상기 b) 단계의 중합 단계를 통해 형성된 중합된 가닥을 c) 단계 이후PCR 반응을 통해 증폭하는 단계를 더욱 포함할 수 있다.Specifically, the method may further include amplifying the polymerized strand formed through the polymerization step of step b) through a PCR reaction after step c).
또한, 구체적으로 상기 타겟 RNA는 스몰(small) RNA 일 수 있다. 상기 스몰 RNA는 miRNA, siRNA 외에도 약 50개 이하의 뉴클레오티드로 이루어진 RNA를 통칭하는 것일 수 있다.Also, specifically, the target RNA may be a small RNA. The small RNA may collectively refer to RNA consisting of about 50 nucleotides or less in addition to miRNA and siRNA.
본 발명에서, 실리카는 규소의 산화물인 이산화규소(SiO2)로서 실리카의 제형은 멤브레인(membrane), 나노입자, 분말 등으로서 필요에 따라 제한없이 적용될 수 있다.In the present invention, silica is silicon dioxide (SiO 2 ), which is an oxide of silicon, and the formulation of silica can be applied without limitation as needed as a membrane, nanoparticles, powder, and the like.
본 발명 일 실시예에서는 종래 DNA 클린업 방식을 적용하는 대신 실리카를 처리한 경우, 단일가닥(single strand, ss) DNA형태인 타겟 RNA에 미결합한 센서 DNA를 효과적으로 제거함으로써 비특이적 검출 반응이 억제됨을 확인하였다.In one embodiment of the present invention, when silica was treated instead of applying the conventional DNA cleanup method, the non-specific detection reaction was inhibited by effectively removing the sensor DNA unbound to the target RNA in the form of single-stranded (ss) DNA. .
또한 구체적으로, 상기 실리카는 pH 7.5 내지 pH 8.5의 조건에서 처리되는 것일 수 있다. In addition, specifically, the silica may be treated under the conditions of pH 7.5 to pH 8.5.
본 발명 일 실시예에서는 실리카가 처리된 경우 pH 7.5 내지 pH 8.5 범위에서 dsDNA는 제거되지 않으면서도 타겟 RNA와 결합되지 않은 센서 DNA(ssDNA)가 제거되어 qPCR 결과가 나타나지 않는 것을 확인하였는 바(도 2), 상기 실리카의 처리는 pH 7.5 내지 pH 8.5 범위에서 ssDNA에 대한 특이적인 제거가 이루어지도록 적용될 수 있다.In one embodiment of the present invention, when silica was treated, dsDNA was not removed in the range of pH 7.5 to pH 8.5, but sensor DNA (ssDNA) not bound to target RNA was removed, confirming that qPCR results did not appear (Fig. 2). ), the treatment of silica can be applied so that specific removal of ssDNA is made in the range of pH 7.5 to pH 8.5.
본 발명의 다른 측면은 타겟 RNA의 상보적인 서열을 포함하는 센서 DNA; 및 실리카를 포함하는, RNA 검출용 조성물에 관한 것이다.Another aspect of the present invention is a sensor DNA comprising a complementary sequence of a target RNA; And it relates to a composition for detecting RNA, comprising silica.
구체적으로, 상기 센서 DNA는 타겟 RNA를 인지하여 혼성화하고, 상기 실리카는 타겟 RNA와 혼성화되지 않은 센서 DNA를 제거하는 것일 수 있다. 또한, 상기 센서 DNA는 단일 가닥(single strand, ss) DNA 형태인 것일 수 있다.Specifically, the sensor DNA may be hybridized by recognizing the target RNA, and the silica may be removing the sensor DNA that is not hybridized with the target RNA. In addition, the sensor DNA may be in the form of single-stranded (ss) DNA.
본 발명 일 실시예에서는 타겟 RNA에 대한 상보적인 서열을 포함하는ssDNA 형태인 센서 DNA를 샘플에 혼합하여 타겟 RNA 검출 반응을 진행하였으며, 상기 검출 반응에 있어서 실리카를 처리한 경우 타겟 RNA와 혼성화되지 않은 미반응 센서 DNA가 효과적으로 제거됨으로써 비특이적 PCR 반응이 억제됨을 확인하였다(도 3 내지 도 9). 이에 따라, 타겟 RNA를 검출하기 위한 센서 DNA 및 실리카를 포함하는 RNA 검출용 조성물은 비특이적 반응을 억제하여 정확도를 향상시킨 검출 반응을 통해 타겟 RNA가 정확히 검출되도록 할 수 있다. In one embodiment of the present invention, a target RNA detection reaction was performed by mixing the sensor DNA in the form of ssDNA containing a sequence complementary to the target RNA to the sample, and when silica was treated in the detection reaction, the target RNA did not hybridize It was confirmed that the non-specific PCR reaction was suppressed by effectively removing the unreacted sensor DNA ( FIGS. 3 to 9 ). Accordingly, the composition for detecting RNA including the sensor DNA and silica for detecting the target RNA can accurately detect the target RNA through a detection reaction with improved accuracy by suppressing a non-specific reaction.
나아가, 본 발명 일 실시예에서는 실리카 처리 단계를 포함한 경우 시판되고 있는 키트를 이용한 종래 DNA 클린업 방식을 적용한 경우에 비해 현저히 낮은 Ct값을 보여 민감도가 현저히 향상되었음을 확인하였다(도 8). 또한, 정량적 검출에 있어서도 실리카를 처리한 경우 종래 DNA 클린업 방식을 적용한 경우에 비해 최소 4 배 내지 300 배 이상의 분자 수를 탐지하였는 바(도 9), 이는 타겟 RNA와 결합되지 않고 남아있는 센서 DNA를 효과적으로 제거하여 비특이적 반응을 현저히 감소시킴으로써 정량적으로도 향상된 검출능을 나타낸 것이다.Furthermore, in one embodiment of the present invention, when the silica treatment step was included, it was confirmed that the sensitivity was significantly improved by showing a significantly lower Ct value compared to the case where the conventional DNA cleanup method using a commercially available kit was applied (FIG. 8). In addition, in the case of quantitative detection, when silica was treated, the number of molecules was detected at least 4 to 300 times higher than when the conventional DNA cleanup method was applied (FIG. 9), which detects the remaining sensor DNA without binding to the target RNA. By effectively removing it, the non-specific reaction was significantly reduced, and thus the detection ability was improved quantitatively.
즉, 본 발명의 실리카 처리 단계를 포함하는 RNA 검출 방법은 종래 엔도뉴클레아제 처리 전후의 DNA 클린업 과정을 거치지 않아 검출 시간을 단축하면서도 비특이적 반응을 제거함으로써 검출 민감도를 현저히 향상시킬 수 있다. That is, the RNA detection method including the silica treatment step of the present invention does not go through the DNA cleanup process before and after the conventional endonuclease treatment, thereby shortening the detection time and eliminating non-specific reactions, thereby remarkably improving the detection sensitivity.
상기 RNA 검출용 조성물은 센서 DNA; 및 실리카 외에도 완충액, PCR 프라이머 등을 더욱 포함할 수 있으며, 추가로 포함되는 구성요소는 필요에 따라 변경하여 적용될 수 있다.The composition for detecting RNA comprises: sensor DNA; And it may further include a buffer, a PCR primer, etc. in addition to silica, and additionally included components can be applied by changing as needed.
본 발명의 또 다른 일 측면은 실리카를 처리하는 단계를 포함하는, 단일가닥 DNA(single strand DNA, ssDNA) 제거 방법에 관한 것이다. 구체적으로 상기 실리카는 pH 7.5 내지 pH 8.5의 조건에서 처리되는 것일 수 있다.Another aspect of the present invention relates to a method for removing single-stranded DNA (ssDNA), comprising the step of treating silica. Specifically, the silica may be treated under the conditions of pH 7.5 to pH 8.5.
본 발명 일 실시예에서는 실리카를 처리하는 경우 타겟 RNA와 결합되지 못한 ssDNA 형태의 센서 DNA가 제거됨을 확인하였는 바, 실리카를 처리하여 ssDNA를 제거하는 방법에 적용할 수 있다.In one embodiment of the present invention, it was confirmed that the sensor DNA in the form of ssDNA that is not bound to the target RNA is removed when silica is treated, so it can be applied to a method of removing ssDNA by treating silica.
특히, 상기 실리카는 pH 7.5 내지 pH 8.5 조건에서 처리될 경우, dsDNA는 제거하지 않고 ssDNA만을 제거하는 특이적 반응을 나타내었는 바, pH 7.5 내지 pH 8.5 조건에서 dsDNA의 손실없이 ssDNA를 특이적으로 제거할 수 있다.In particular, when the silica was treated at pH 7.5 to pH 8.5, it showed a specific reaction of removing only ssDNA without removing dsDNA, and specifically removing ssDNA without loss of dsDNA at pH 7.5 to pH 8.5. can do.
본 발명의 RNA 검출 방법 및 검출에 사용되는 센서 DNA의 구성은 검출 한계가 아토몰(amol) 수준으로 매우 낮은 바, 종래 RNA 검출 기술에 비해 민감도 및 정확도가 현저히 우수하다. 특히, 실리카를 처리함으로써 비특이적 반응을 억제하면서도 검출 민감도를 현저히 향상시켰으며, 종래의 DNA 클린업 방식을 적용한 검출 방법에 비해 검출 단계가 감소되어 검출에 소요되는 시간을 단축시킬 수 있다.Since the detection limit of the RNA detection method of the present invention and the configuration of the sensor DNA used for detection is very low at the level of an amol, the sensitivity and accuracy are significantly superior to those of the conventional RNA detection technology. In particular, by treating silica, the detection sensitivity was significantly improved while suppressing non-specific reactions, and the detection step was reduced compared to the detection method using the conventional DNA cleanup method, thereby shortening the time required for detection.
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.It should be understood that the effects of the present invention are not limited to the above-described effects, and 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 종래 DNA 클린업 방식 적용 및 본 발명의 실리카 처리 단계 적용시의 반응 순서도를 나타낸 것이다. 실리카 처리의 경우 검출에 필요한 단계가 감소되어 소요 시간이 감축된다.
도 2는 pH에 따른 실리카의 ssDNA 센서, dsDNA 제거 효과를 비교한 결과를 나타낸 것이다.
도 3은 전혈에서의 종래 DNA 클린업 방식 및 실리카 처리시의 ssDNA 센서 제거 효과를 비교한 결과를 나타낸 것이다(-RNA: 타겟 RNA와 미결합된 센서 DNA(ssDNA), +RNA: 타겟 RNA와 결합된 센서 DNA(double strand 형성); 3A: hsa-miR486-5p, 3B: hsa-miR210-3p).
도 4는 혈장(plasma)에서의 종래 DNA 클린업 방식 및 실리카 처리시의 ssDNA 센서 제거 효과를 비교한 결과를 나타낸 것이다(-RNA: 타겟 RNA와 미결합된 센서 DNA(ssDNA), +RNA: 타겟 RNA와 결합된 센서 DNA(double strand 형성)).
도 5 내지 도 7은 멀티플렉스 검출시 비특이적 반응 감소 효과를 비교한 결과를 나타낸 것이다(-RNA: 타겟 RNA와 미결합된 센서 DNA(ssDNA), +RNA: 타겟 RNA와 결합된 센서 DNA(double strand 형성); 5A: hsa-miR486-5p, 5B: hsa-miR1290, 6A: hsa-miR132-3p, 6B: hsa-miR146a-5p, 7A: hsa-miR27a-3p, 7B: hsa-miR31-5p).
도 8은 종래 클린업 방식 적용 및 본 발명의 실리카 처리 단계 적용에 따른 검출 민감도를 Ct값을 통해 확인한 결과를 나타낸 것이다.
도 9는 종래 클린업 방식 적용 및 본 발명의 실리카 처리 단계 적용에 따른 검출 민감도를 qPCR standard curve를 이용한 분자 개수 환산을 통해 확인한 결과를 나타낸 것이다. 1 shows a reaction flow diagram when applying the conventional DNA cleanup method and applying the silica treatment step of the present invention. In the case of silica treatment, the steps required for detection are reduced, thus reducing the time required.
Figure 2 shows the results of comparing the ssDNA sensor, dsDNA removal effect of silica according to pH.
3 shows the results of comparing the ssDNA sensor removal effect of the conventional DNA cleanup method and silica treatment in whole blood (-RNA: sensor DNA unbound with target RNA (ssDNA), +RNA: combined with target RNA Sensor DNA (double strand formation); 3A: hsa-miR486-5p, 3B: hsa-miR210-3p).
4 shows the results of comparing the ssDNA sensor removal effect during the conventional DNA cleanup method and silica treatment in plasma (-RNA: target RNA and unbound sensor DNA (ssDNA), +RNA: target RNA combined with sensor DNA (forming a double strand).
5 to 7 show the results of comparison of the effect of reducing the non-specific reaction during multiplex detection (-RNA: sensor DNA unbound to target RNA (ssDNA), +RNA: sensor DNA bound to target RNA (double strand) 5A: hsa-miR486-5p, 5B: hsa-miR1290, 6A: hsa-miR132-3p, 6B: hsa-miR146a-5p, 7A: hsa-miR27a-3p, 7B: hsa-miR31-5p).
8 shows the results of checking the detection sensitivity through the Ct value according to the application of the conventional cleanup method and the application of the silica treatment step of the present invention.
9 shows the result of confirming the detection sensitivity according to the application of the conventional clean-up method and the application of the silica treatment step of the present invention through conversion of the number of molecules using the qPCR standard curve.
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 way of Examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.
실시예 1. pH에 따른 실리카의 단일가닥 DNA(ssDNA) 특이적 제거 효과 확인Example 1. Confirmation of single-stranded DNA (ssDNA) specific removal effect of silica according to pH
인간 혈액 샘플로부터 XENOPURE RNA PURIFICATION KIT (whole blood) (cat. No. 93667873-WB)을 사용하여 RNA를 정제한 후 100 ng의 전혈 RNA와 타겟 RNA 인 hsa-miR486-5p의 탐지 센서 DNA(ssDNA) 500 amol를 혼합하였다.After RNA purification from human blood samples using XENOPURE RNA PURIFICATION KIT (whole blood) (cat. No. 93667873-WB), 100 ng of whole blood RNA and a detection sensor DNA (ssDNA) of hsa-miR486-5p, a target RNA 500 amol was mixed.
상기 센서 DNA는 타겟 RNA와의 상보적인 서열을 포함하는 센싱 영역을 포함하는 단일가닥 DNA(ssDNA)로서, 상기 센싱 영역은 타겟 RNA를 감지하여 타겟 RNA와 혼성화하는 것으로서, '센서 DNA'로 명명하였다. 타겟 RNA와 혼성화되지 않은 센서 DNA는 ssDNA로 남아 검출에 있어 비특이적 반응을 일으킬 수 있는 바, 검출을 위한 PCR 반응 전에 이를 제거하는 단계가 필요하다. 본 발명에서는 실리카를 혼합하여 센서 DNA에 의한 비특이적 반응을 억제시킬 수 있음을 확인하였다.The sensor DNA is a single-stranded DNA (ssDNA) including a sensing region including a sequence complementary to a target RNA. The sensing region detects the target RNA and hybridizes with the target RNA, and was named 'sensor DNA'. Since the sensor DNA that is not hybridized with the target RNA remains as ssDNA and may cause a non-specific reaction in detection, it is necessary to remove it before the PCR reaction for detection. In the present invention, it was confirmed that non-specific reactions caused by sensor DNA can be suppressed by mixing silica.
타겟 RNA와 센서 DNA가 혼성화되도록 한 후, 타겟 RNA를 주형으로 하고, 센서 DNA를 프라이머로 하여, 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, 25°C)), 1 ㎕의 10 mM dNTP 및 DNA 중합효소(XenoT-PoL)를 95 ℃에서 1분 간 가열한 후 63 ℃에서 5 분간 인큐베이션(incubation)하였다.After allowing the target RNA and the sensor DNA to hybridize, using the target RNA as a template and the sensor DNA as a primer, 2 μl of reaction buffer (reaction Buffer, 200 mM Tris-HCl, 100 mM (NH 4 ) 2 SO 4 , 100 mM KCl, 20 mM MgSO 4 , 1% Triton X-100, (pH 8.8, 25 °C)), 1 µl of 10 mM dNTP and DNA polymerase (XenoT-PoL) at 95 °C for 1 min. After heating, it was incubated at 63 °C for 5 minutes.
이후, 중합된 가닥을 증폭하는 qPCR 수행 전 별도의 DNA 클린업 과정 없이 pH를 달리하여 실리카 처리 여부에 따른 ssDNA 제거능을 확인하였다.Thereafter, before performing qPCR to amplify the polymerized strand, the pH was changed without a separate DNA cleanup process, and the ability to remove ssDNA according to silica treatment was confirmed.
각각의 샘플에 실리카(Xenohelix, Cat No. 9366-745422) 30 ug을 혼합한 후 5 mM Tris-HCl, 2.5 mM (NH4)2SO4, 2.5 mM KCl, 0.5 mM MgSO4, 0.025% Triton®X-100 조건에서, 각각 pH가 5.5, 7.5, 8.5, 9.5 및 10.5가 되도록 한 후 5 분 동안 25 ℃에서 볼텍싱(vortexing)하여 혼합하였다.After mixing 30 ug of silica (Xenohelix, Cat No. 9366-745422) in each sample, 5 mM Tris-HCl, 2.5 mM (NH 4 ) 2 SO 4 , 2.5 mM KCl, 0.5 mM MgSO 4 , 0.025% Triton® In the X-100 condition, the pH was adjusted to 5.5, 7.5, 8.5, 9.5 and 10.5, respectively, and then mixed by vortexing at 25° C. for 5 minutes.
이후 상기와 같이 pH를 달리하여 제조한 각 혼합액을 DNA 컬럼(Xenohelix, Cat No. 9366-3622656B) 으로 옮겨 1분간 6,000 rpm으로 원심분리하여 받은 분리액에 증류수를 첨가하여 200 ㎕로 용리(elution)하였다. 이 중, 3 ul 샘플을 hsa-miR486-5p 센서 특이적 프라이머를 이용하여 각각 95 ℃ 3 분 1사이클, 95 ℃ 10초, 64 ℃ 30초 40 사이클 조건으로 qPCR을 수행하였다.Then, each mixed solution prepared by changing the pH as described above was transferred to a DNA column (Xenohelix, Cat No. 9366-3622656B) and centrifuged at 6,000 rpm for 1 minute. did. Of these, 3 ul samples were subjected to qPCR using the hsa-miR486-5p sensor-specific primer under the conditions of 95 ° C for 3 min 1 cycle, 95 ° C for 10 sec, and 64 ° C for 30
그 결과, 도 2에 나타난 바와 같이 pH 5.5 조건에서는 타겟 RNA 부존재에 따라 남은 센서 DNA(ssDNA) 및 타겟 RNA와 센서 DNA의 결합 및 중합반응에 따라 생성된 dsDNA(double strand DNA)가 모두 실리카 처리에 의해 제거되었다. As a result, as shown in FIG. 2, under the pH 5.5 condition, the remaining sensor DNA (ssDNA) and dsDNA (double stranded DNA) generated by the binding and polymerization of the target RNA and the sensor DNA were all treated with silica. was removed by
그러나, pH 7.5~8.5 조건에서는 실리카 처리에 의해 타겟 RNA와 결합되지 않은 센서 DNA(ssDNA)는 제거되어 qPCR 결과가 나타나지 않은 반면, 타겟 RNA의 존재에 따라 센서 DNA가 타겟 RNA에 결합하고 중합반응에 따라 생성된 dsDNA는 제거되지 않고 보존된 것을 확인하였다. 반면, pH 9.5 및 10.5 조건에서는 ssDNA의 제거효과가 감소되었다.However, under the conditions of pH 7.5 to 8.5, qPCR results did not appear because sensor DNA (ssDNA) not bound to target RNA was removed by silica treatment. It was confirmed that the generated dsDNA was not removed and was preserved. On the other hand, the removal effect of ssDNA was reduced at pH 9.5 and 10.5 conditions.
상기와 같은 결과로부터 실리카 처리에 의해 ssDNA가 제거될 수 있으며, 특히 pH 7.5~8.5에서는 ssDNA가 dsDNA와 구분되어 특이적으로 제거됨으로써 타겟 RNA와 결합되지 않고 남아있는 ssDNA 형태의 센서 DNA들만 간편하고 빠른 시간에 제거되는 것을 확인하였다. 이는 본 발명에서 타겟 RNA를 검출함에 있어서 종래 중합반응 이후 DNA 클린업 과정을 거친 후 다시 엔도뉴클레아제 처리 후 DNA 클린업 과정을 거친 후에 PCR 반응이 진행된 것과 달리 DNA 클린업 과정 없이 엔도뉴클레아제 처리 후 실리카를 처리하는 것만으로도 노이즈를 일으킬 수 있는 미결합 센서 DNA를 제거할 수 있다는 점에서 클린업 단계 축소에 따른 검출시간 감소, 미결합 센서 DNA를 특이적으로 제거 가능함에 따른 부정확성을 감소시키는 장점이 있음을 확인한 것이다.From the above results, ssDNA can be removed by silica treatment. In particular, at pH 7.5 to 8.5, ssDNA is separated from dsDNA and specifically removed, so that only the sensor DNA in the form of ssDNA remaining without binding to the target RNA is simple and quick. It was confirmed that it was removed over time. In the present invention, in detecting target RNA, unlike the conventional polymerization reaction, DNA cleanup process, endonuclease treatment, and PCR reaction after DNA cleanup process, silica after endonuclease treatment without DNA cleanup process In that unbound sensor DNA that can cause noise can be removed just by treating will confirm
실시예 2. 전혈(whole blood)에서의 실리카의 ssDNA 제거 효과 확인Example 2. Confirmation of ssDNA removal effect of silica in whole blood
인간 혈액 샘플로부터 XENOPURE RNA PURIFICATION KIT (whole blood) (cat. No. 93667873-WB)을 사용하여 RNA를 정제하였다. 이후 100 ng의 전혈 RNA와 타겟 RNA 인 hsa-miR486-5p 및 hsa-miR210-3p 각각의 탐지 센서 DNA(ssDNA) 500 amol씩 혼합하고, 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 하였다. RNA was purified from human blood samples using XENOPURE RNA PURIFICATION KIT (whole blood) (cat. No. 93667873-WB). Then, 100 ng of whole blood RNA and 500 amol of detection sensor DNA (ssDNA) of each of the target RNAs, hsa-miR486-5p and hsa-miR210-3p, were mixed, and 2 μl of reaction buffer (reaction Buffer, 200 mM Tris- HCl, 100 mM (NH 4 ) 2 SO 4 , 100 mM KCl, 20 mM MgSO 4 , 1% Triton X-100, (pH 8.8 at 25 °C)), 1 μl of 10 mM dNTP and 2 units of DNA polymer Rase (XenoT-POL) was mixed. The mixture was heated at 95 °C for 1 minute and then incubated at 63 °C for 5 minutes.
상기 혼합액을 엔도뉴클레아제 처리 후 종래의 DNA 클린업을 진행하는 샘플, 엔도뉴클레아제 처리 후 실리카를 처리하는 샘플로 나누어 진행하였다.The mixture was divided into a sample subjected to conventional DNA cleanup after endonuclease treatment, and a sample treated with silica after endonuclease treatment.
구체적으로, 혼합액을 20 ㎕씩 나눈 후 이후 종래의 DNA 클린업을 진행하는 샘플에는 시판되고 있는 MEGAquick-spin™Total Fragment DNA Purification Kit를 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 뉴클레아제(nuclease))을 섞어주고 30 °C에서 10 분간 인큐베이션 하였다. 이후 다시 MEGAquick-spin™Total Fragment DNA Purification Kit을 이용하여 클린업하고, 200 ㎕의 증류수로 용리한 후 3 ul 샘플을 hsa-miR486-5p, hsa-miR210-3p 센서 특이적 프라이머를 이용하여 95 ℃ 3 분 1사이클, 95 ℃ 10초, 64 ℃ 30초 45 사이클조건으로 qPCR을 수행하였다.Specifically, after dividing the mixture by 20 μl, the samples subjected to conventional DNA cleanup are cleaned up using a commercially available MEGAquick-spin™ Total Fragment DNA Purification Kit, eluted with 40 μl distilled water, and endonuclease The mixture (40 mM sodium acetate (sodium acetate, pH 4.5 at 25 °C), 300 mM NaCl, 2 mM ZnSO 4 and 200 unit nuclease) was mixed and incubated at 30 °C for 10 minutes. After that, it was cleaned up again using the MEGAquick-spin™ Total Fragment DNA Purification Kit, eluted with 200 μl of distilled water, and then the 3 μl sample was washed at 95° C. 3 using hsa-miR486-5p, hsa-miR210-3p sensor-specific primers. qPCR was performed under the conditions of 1 min cycle, 95 ℃ 10 sec, 64 ℃ 30
이와 달리, 본 발명의 실리카를 처리하는 단계를 포함하는 방법에 있어서는 엔도뉴클레아제 처리 전 별도의 클린업 과정 없이 20 ㎕의 샘플을 엔도뉴클레아제 혼합물 (5 mM Tris-HCl, 2.5 mM (NH4)2SO4, 2.5 mM KCl, 10.5 mM MgSO4, 2 mM ZnSO4, 0.025% Triton®X-100, pH 6.0) 및 20 unit 엔도뉴클레아제(endonuclease))을 섞어주고 60 °C에서 5 분간 인큐베이션 하였다. 이후 실리카 30 ug을 섞어준 후 2.5 mM Tris-HCl, 1.25 mM (NH4)2SO4, 1.25 mM KCl, 5.25 mM MgSO4, 0.0125% Triton®X-100 조건에서 5 분간 25 ℃에서 볼텍싱하였다. 이후 혼합액을 DNA 컬럼으로 옮겨 1 분간 6,000 rpm으로 원심분리하여 받은 분리액에 증류수를 첨가하여 200 ㎕로 용리하였다. 이 중, 3 ul 샘플을 hsa-miR486-5p, hsa-miR210-3p 센서 특이적 프라이머를 이용하여 각각 95 ℃3 분 1사이클, 95 ℃10초, 64 ℃30초 45 사이클 조건으로 qPCR을 수행하였으며, qPCR 수행 전 별도의 클린업 과정은 거치지 않았다.In contrast, in the method comprising the step of treating the silica of the present invention, 20 μl of the sample was prepared with an endonuclease mixture (5 mM Tris-HCl, 2.5 mM (NH 4 ) 2 SO 4 , 2.5 mM KCl, 10.5 mM MgSO 4 , 2 mM ZnSO 4, 0.025% Triton®X-100, pH 6.0) and 20 unit endonuclease) were mixed and heated at 60 °C for 5 min. incubated. After mixing 30 ug of silica, 2.5 mM Tris-HCl, 1.25 mM (NH 4 ) 2 SO 4 , 1.25 mM KCl, 5.25 mM MgSO 4 , 0.0125% Triton®X-100 was vortexed for 5 minutes at 25 ° C. . Then, the mixture was transferred to a DNA column, centrifuged at 6,000 rpm for 1 minute, distilled water was added to the obtained separation solution, and 200 μl was eluted. Of these, 3 ul samples were subjected to qPCR using hsa-miR486-5p and hsa-miR210-3p sensor-specific primers under the conditions of 95 ℃ 3 min 1 cycle, 95 ℃ 10 sec, and 64 ℃ 30
그 결과, 도 3에 나타난 바와 같이 종래의 DNA 클린업 과정 보다 실리카 처리시 타겟 RNA와 미결합된 센서 DNA(ssDNA)의 특이적인 제거가 더 많이 이루어져 qPCR 수행시 더 높은 Ct 값이 나타남을 확인하였다(-RNA: 타겟 RNA와 미결합된 센서 DNA(ssDNA), +RNA: 타겟 RNA와 결합된 센서 DNA(double strand 형성); 3A: hsa-miR486-5p, 3B: hsa-miR210-3p). 또한, 센서 DNA를 달리하여 적용하더라도 동일하게 실리카 처리시 비특이적 반응이 더 많이 제거됨을 확인하였다.As a result, as shown in FIG. 3 , more specific removal of target RNA and unbound sensor DNA (ssDNA) was performed during silica treatment than in the conventional DNA cleanup process, and it was confirmed that a higher Ct value was displayed during qPCR ( -RNA: sensor DNA unbound with target RNA (ssDNA), +RNA: sensor DNA bound with target RNA (double strand formation); 3A: hsa-miR486-5p, 3B: hsa-miR210-3p). In addition, it was confirmed that even if the sensor DNA was applied differently, more non-specific reactions were removed during the same silica treatment.
실시예 3. 혈장(plasma)에서의 실리카의 ssDNA 제거 효과 확인Example 3. Confirmation of ssDNA removal effect of silica in plasma
250 ㎕의 인간 혈장 샘플에서 XENOPURE RNA PURIFICATION KIT (Plasma/Serum) (cat. No. 93667873-SP)를 사용하여 RNA를 정제하였다. 상기 정제된 RNA를 40 ㎕ Elution 버퍼에 용리한 후, 13 ㎕ 샘플에 hsa-miR210-3p 센서 DNA를 500 amol 혼합한 후 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, 25°C)), 1 ㎕의 10 mM dNTP 및 2 unit DNA 폴리머라제(XenoT-POL)를 넣어준 후 95 ℃에서 1분 간 가열한 후 63 ℃에서 5 분간 인큐베이션 하였다.RNA was purified from 250 μl human plasma samples using the XENOPURE RNA PURIFICATION KIT (Plasma/Serum) (cat. No. 93667873-SP). After eluting the purified RNA in 40 μl Elution buffer, 500 amol of hsa-miR210-3p sensor DNA was mixed with 13 μl sample and 2 μl of reaction buffer (reaction Buffer, 200 mM Tris-HCl, 100 mM) (NH 4 ) 2 SO 4 , 100 mM KCl, 20 mM MgSO 4 , 1% Triton X-100, (pH 8.8, 25 °C)), 1 μl of 10 mM dNTP and 2 unit DNA polymerase (XenoT-POL) ), heated at 95 °C for 1 minute, and incubated at 63 °C for 5 minutes.
상기 혼합액을 엔도뉴클레아제 처리 후 종래의 DNA 클린업을 진행하는 샘플, 엔도뉴클레아제 처리 후 실리카를 처리하는 샘플로 나누어 진행하였으며, 이하 세부 진행 과정은 상기 실시예 2와 같다.The mixed solution was divided into a sample subjected to conventional DNA cleanup after endonuclease treatment and a sample treated with silica after endonuclease treatment, and the detailed procedure is the same as in Example 2.
그 결과, 도 4에 나타난 바와 같이 혈장 샘플을 이용한 경우에도 실리카를 처리한 경우 타겟 RNA와 미결합된 센서 DNA의 특이적인 제거가 더 많이 이루어져 qPCR 수행시 더 높은 Ct 값이 나타남을 확인하였다. 특히, 센서 DNA가 타겟 RNA에 결합하고 중합 반응에 따라 생성된 dsDNA의 경우, 실리카 처리시 Ct 값이 더 낮게 나타나 검출 대상의 손실이 감소하는 효과까지 나타남을 확인하였다.As a result, as shown in FIG. 4 , even when plasma samples were used, it was confirmed that, when silica was treated, the specific removal of sensor DNA unbound to the target RNA was increased, resulting in a higher Ct value during qPCR. In particular, it was confirmed that, in the case of dsDNA produced according to the polymerization reaction after the sensor DNA binds to the target RNA, the Ct value is lower when silica is treated, thereby reducing the loss of the detection target.
즉, 실리카 처리 단계를 포함한 본 발명의 검출 방법은 특히 혈장 내 스몰(small) RNA 탐지에 있어 종래의 DNA 클린업 단계를 포함하는 것에 비해 효율적으로 ssDNA인 센서 DNA를 제거하면서도 검출 대상이 되는 타겟 RNA-센서 DNA 결합에 따라 중합된 dsDNA의 손실을 감소시켜 검출의 정확도 및 민감도를 향상시킬 수 있다.That is, the detection method of the present invention including the silica treatment step effectively removes the sensor DNA, which is ssDNA, as compared to the conventional DNA cleanup step, especially in the detection of small RNA in plasma, the target RNA- By reducing the loss of polymerized dsDNA upon binding of the sensor DNA, the accuracy and sensitivity of detection can be improved.
실시예 4. 멀티플렉스 검출시 비특이적 반응 감소 효과 비교Example 4. Comparison of the effect of reducing non-specific response upon detection of multiplex
종래의 DNA 클린업과 실리카 처리 각각에 대하여 여러 종류의 센서 DNA 및 타겟 RNA를 혼합하여 멀티플렉스 검출시의 비특이적 검출 반응 감소 효과를 비교하였다.For each of the conventional DNA cleanup and silica treatment, various types of sensor DNA and target RNA were mixed to compare the effect of reducing non-specific detection reaction during multiplex detection.
구체적으로, 인간 혈액 샘플에서 RNA 정제 후 100 ng의 전혈 RNA와 6 종의 miRNA(hsa-miR486-5p, hsa-miR1290, hsa-miR132-3p, hsa-miR146a-5p, hsa-miR27a-3p 및 hsa-miR31-5p) 검출을 위한 각각의 센서 DNA 6종을 각 500 amol씩 혼합하였다.Specifically, after RNA purification from human blood samples, 100 ng of whole blood RNA and 6 miRNAs (hsa-miR486-5p, hsa-miR1290, hsa-miR132-3p, hsa-miR146a-5p, hsa-miR27a-3p and hsa) -miR31-5p) each of 6 types of sensor DNA for detection was mixed by 500 amol each.
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, 25°C)), 1 ㎕의 10 mM dNTP 및 2 unit DNA 폴리머라제(XenoT-POL)를 넣어준 후 95 ℃에서 1분 간 가열한 후 63 ℃에서 5 분간 인큐베이션 하였다.Then, 2 μl of reaction buffer (reaction Buffer, 200 mM Tris-HCl, 100 mM (NH 4 ) 2 SO 4 , 100 mM KCl, 20 mM MgSO 4 , 1% Triton X-100, (pH 8.8, 25) °C)), 1 μl of 10 mM dNTP and 2 unit DNA polymerase (XenoT-POL) were added, heated at 95 °C for 1 minute, and incubated at 63 °C for 5 minutes.
상기 혼합액을 분리하여 엔도뉴클레아제를 처리한 후, 하나의 샘플은 종래 DNA 클린업 방식을 적용하여 진행하고, 다른 하나의 샘플은 실리카 처리 후 각각 qPCR을 진행하였다. 세부 진행 과정은 상기 실시예 2와 같다.After the mixture was separated and treated with endonuclease, one sample was processed by applying the conventional DNA cleanup method, and the other sample was subjected to qPCR after silica treatment. The detailed process is the same as in Example 2.
그 결과 도 5 내지 도 7에 나타난 바와 같이 멀티플렉스 방식으로 6 종에 대한 타겟 RNA를 동시에 검출하는 경우에도, 실리카를 처리한 경우 RNA와 미결합된 센서 DNA의 제거 효과가 종래 DNA 클린업 방식을 적용한 경우에 비해 현저히 우수함을 확인하였다.As a result, as shown in FIGS. 5 to 7, even when the target RNA for 6 species is simultaneously detected in the multiplex method, the removal effect of the sensor DNA unbound to the RNA when the silica is treated was applied to the conventional DNA cleanup method. It was confirmed that it was significantly superior to the case.
구체적으로, hsa-miR486-5p(도 5A), hsa-miR1290(도 5B), hsa-miR146a-5p(도 6B) 및 hsa-miR31-5p(도 7B)에 대해서는 실리카 처리에 의해 타겟 RNA와 결합되지 않은 센서 DNA(ssDNA)가 완전히 제거되어 타겟 RNA와 결합되지 않은 센서 DNA(ssDNA)는 제거되어 qPCR 결과가 나타나지 않은 반면, 타겟 RNA의 존재에 따라 센서 DNA가 타겟 RNA에 결합하고 중합반응에 따라 생성된 dsDNA는 제거되지 않고 보존된 것을 확인하였다. 그러나, 종래의 DNA 클린업 방식을 적용한 경우에는 센서 DNA가 다량 남아있음을 확인하였다.Specifically, for hsa-miR486-5p (FIG. 5A), hsa-miR1290 (FIG. 5B), hsa-miR146a-5p (FIG. 6B) and hsa-miR31-5p (FIG. 7B) by silica treatment, binding to target RNA Sensor DNA (ssDNA) that was not bound to the target RNA was completely removed so that the qPCR result was not shown, whereas the sensor DNA binds to the target RNA depending on the presence of the target RNA and depends on the polymerization reaction. It was confirmed that the generated dsDNA was preserved without being removed. However, it was confirmed that a large amount of sensor DNA remained when the conventional DNA cleanup method was applied.
또한, hsa-miR132-3p(도 6A) 및 hsa-miR27a-3p(도 7A)에 대해서도 종래의 DNA 클린업 과정 보다 실리카 처리시 타겟 RNA와 미결합된 센서 DNA(ssDNA)의 특이적인 제거가 더 많이 이루어져 qPCR 수행시 더 높은 Ct 값이 나타남을 확인하였다.In addition, for hsa-miR132-3p (Fig. 6A) and hsa-miR27a-3p (Fig. 7A), the specific removal of the sensor DNA (ssDNA) unbound to the target RNA was higher during silica treatment than the conventional DNA cleanup process. It was confirmed that a higher Ct value appeared when qPCR was performed.
나아가, 본 멀티플렉스 검출에 있어서도 실리카 처리시 Ct 값이 더 낮게 나타나 검출 대상이 되는 타겟 RNA-센서 DNA 결합에 따라 중합된 dsDNA의 손실이 종래 DNA 클린업 방식을 적용한 것에 비해 현저히 감소되었으며, 이로부터 멀티플렉스 검출에 있어서도 검출의 정확도 및 민감도를 향상시킬 수 있음을 확인하였다. Furthermore, even in this multiplex detection, the Ct value was lower when treated with silica, so that the loss of polymerized dsDNA according to the target RNA-sensor DNA binding to be detected was significantly reduced compared to applying the conventional DNA cleanup method. It was confirmed that the accuracy and sensitivity of detection can be improved even in plex detection.
상기와 같은 결과는 실리카를 처리함으로써 타겟 RNA와 혼성화하지 못하여 비특이적 반응을 일으킬 수 있는 센서 DNA를 효과적으로 제거하여 비특이적 반응에 의한 결과가 검출 결과에 포함되지 않도록 함을 의미한다. 특히, 종래의 엔도뉴클레아제 처리 전후의 DNA 클린업 과정을 거치지 않아 검출 시간을 단축하면서도 비특이적 반응을 최소화함으로써 검출의 정확도를 현저히 향상시킬 수 있음을 나타낸다.The above results mean that the result of the non-specific reaction is not included in the detection result by effectively removing the sensor DNA that can not hybridize with the target RNA and cause a non-specific reaction by treating the silica. In particular, it shows that the detection accuracy can be significantly improved by minimizing the non-specific reaction while shortening the detection time by not going through the DNA cleanup process before and after the conventional endonuclease treatment.
실시예 5. 실리카 처리에 따른 검출 민감도 향상 효과 확인Example 5. Confirmation of the effect of improving detection sensitivity by silica treatment
인간 혈액 샘플로부터 XENOPURE RNA PURIFICATION KIT (whole blood) (cat. No. 93667873-WB)을 사용하여 RNA를 정제하였다. 각각 100, 50, 25, 12.5 ng의 전혈 RNA 및 500 amol의 hsa-miR92a-3p 및 hsa-miR27a-3p 각 센서 DNA를 혼합한 후 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, 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 하였다. RNA was purified from human blood samples using XENOPURE RNA PURIFICATION KIT (whole blood) (cat. No. 93667873-WB). After mixing 100, 50, 25, 12.5 ng of whole blood RNA and 500 amol of each sensor DNA of hsa-miR92a-3p and hsa-miR27a-3p, 2 μl of reaction buffer (200 mM Tris-HCl) , 100 mM (NH 4 ) 2 SO 4 , 100 mM KCl, 20 mM MgSO 4 , 1% Triton X-100, (pH 8.8, 25 °C)), 1 μl of 10 mM dNTP and 2 units of DNA polymerase (XenoT-POL) was mixed. The mixture was heated at 95 °C for 1 minute and then incubated at 63 °C for 5 minutes.
상기 혼합액을 분리하여 엔도뉴클레아제를 처리한 후, 하나의 샘플은 종래 DNA 클린업 방식을 적용하여 진행하고, 다른 하나의 샘플은 실리카 처리 후 각각 qPCR을 진행하였다. 세부 진행 과정은 상기 실시예 2와 같다.After the mixture was separated and treated with endonuclease, one sample was processed by applying the conventional DNA cleanup method, and the other sample was subjected to qPCR after silica treatment. The detailed process is the same as in Example 2.
그 결과, 도 8에 나타난 바와 같이, 실리카를 처리한 경우 종래의 DNA 클린업 방식을 적용한 것에 비해 검출능에서 현저히 향상된 민감도를 보여 hsa-miR27a-3p(도 8A) 및 hsa-miR92a-3p(도 8B) 모두에 대해 100, 50, 25, 12.5 ng에서 더 낮은 Ct값이 나타남을 확인하였다.As a result, as shown in FIG. 8, when silica was treated, it showed significantly improved sensitivity in detection compared to applying the conventional DNA cleanup method, and hsa-miR27a-3p (FIG. 8A) and hsa-miR92a-3p (FIG. 8B ), it was confirmed that lower Ct values appeared at 100, 50, 25, and 12.5 ng for all of them.
또한, 도 9에 나타난 바와 같이, hsa-miR92a-3p 및 hsa-miR27a-3p 분자 개수에 대한 qPCR standard curve를 이용하여 각 샘플에서 ㎕당 hsa-miR92a-3p 및 hsa-miR27a-3p 각각의 분자 개수를 환산한 결과, 실리카를 처리한 샘플은 종래의 DNA 클린업 방식을 적용한 샘플보다 8.89~367.15 배의 hsa-miR27a-3p(도 9A), 4.28~106.14 배의 hsa-miR92a-3p(도 9B)를 탐지할 수 있음을 확인하였다.In addition, as shown in FIG. 9 , the number of molecules of each hsa-miR92a-3p and hsa-miR27a-3p per μl in each sample using the qPCR standard curve for the number of hsa-miR92a-3p and hsa-miR27a-3p molecules As a result of converting It was confirmed that it could be detected.
즉, 본 발명의 실리카 처리 단계를 포함하는 RNA 검출 방법은 종래의DNA 클린업 방식을 적용한 것에 비해 비특이적 반응을 제거함으로써 검출 민감도를 현저히 향상시켜 정량적으로도 향상된 검출능을 나타낼 수 있다.That is, the RNA detection method including the silica treatment step of the present invention can significantly improve detection sensitivity by eliminating non-specific reactions compared to applying the conventional DNA cleanup method, thereby exhibiting quantitatively improved detection ability.
상기 실시예 1 내지 5의 각 타겟 RNA 검출을 위한 센서 DNA의 서열은 하기 표 1에 정리된 바와 같다.The sequence of the sensor DNA for each target RNA detection of Examples 1 to 5 is summarized in Table 1 below.
또한, qPCR 수행시 이용된 프라이머 서열은 하기 표 2에 정리된 바와 같다.In addition, the primer sequences used in performing qPCR are as summarized in Table 2 below.
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 foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and likewise 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 한다The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.
<110> Xenohelix. Co., Ltd. <120> A METHOD OF DETECTING TARGET RNA COMPRISING SILICA TREATMENT <130> 20PP31103 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR486-5p <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 for detecting hsa-miR210-3p <400> 2 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagata catcggtgac 60 tggagttgct tggctctggt gtattggttc agccgctgtc acacgcacag 110 <210> 3 <211> 107 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR1290 <400> 3 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt cattcgtgac 60 tggagttgct tggctctggt gtattggttc cctgatccaa aaatcca 107 <210> 4 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR132-3p <400> 4 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatg gcaacgtgac 60 tggagttgct tggctctggt gtattggtcg accatggctg tagactgtta 110 <210> 5 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR146a-5p <400> 5 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt agcttgtgac 60 tggagttgct tggctctggt gtattggtaa cccatggaat tcagttctca 110 <210> 6 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR27a-3p <400> 6 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatc actcagtgac 60 tggagttgct tggctctggt gtattggtgc ggaacttagc cactgtgaa 109 <210> 7 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR31-5p <400> 7 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt acagcgtgac 60 tggagttgct tggctctggt gtattggtag ctatgccagc atcttgcct 109 <210> 8 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR92a-3p <400> 8 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagata agtcagtgac 60 tggagttgct tggctctggt gtattggtac aggccgggac aagtgcaata 110 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR486-5p <400> 9 cgctacagtc gcatacgaga tttaggc 27 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR486-5p <400> 10 tcctgtactg agctgccccg ag 22 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR210-3p <400> 11 ctacagtcgc atacgagata catcg 25 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR210-3p <400> 12 ctgtgcgtgt gacagcggct ga 22 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR1290 <400> 13 gctacagtcg catacgagat tcattc 26 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR1290 <400> 14 tggatttttg gatcagggaa ccaa 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR132-3p <400> 15 gcatacgaga tggcaacgtg 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR132-3p <400> 16 taacagtcta cagccatggt cg 22 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR146a-5p <400> 17 cagtcgcata cgagattagc ttg 23 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR146a-5p <400> 18 tgagaactga attccatggg ttac 24 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR27a-3p <400> 19 gcatacgaga tcactcagtg ac 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR27a-3p <400> 20 ttcacagtgg ctaagttccg c 21 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR31-5p <400> 21 cagtcgcata cgagattaca gc 22 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR31-5p <400> 22 aggcaagatg ctggcatagc t 21 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR92a-3p <400> 23 tcgcatacga gataagtcag tg 22 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR92a-3p <400> 24 tattgcactt gtcccggcct 20 <110> Xenohelix. Co., Ltd. <120> A METHOD OF DETECTING TARGET RNA COMPRISING SILICA TREATMENT <130> 20PP31103 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR486-5p <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 for detecting hsa-miR210-3p <400> 2 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagata catcggtgac 60 tggagttgct tggctctggt gtattggttc agccgctgtc acacgcacag 110 <210> 3 <211> 107 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR1290 <400> 3 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt cattcgtgac 60 tggagttgct tggctctggt gtattggttc cctgatccaa aaatcca 107 <210> 4 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR132-3p <400> 4 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatg gcaacgtgac 60 tggagttgct tggctctggt gtattggtcg accatggctg tagactgtta 110 <210> 5 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR146a-5p <400> 5 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt agcttgtgac 60 tggagttgct tggctctggt gtattggtaa cccatggaat tcagttctca 110 <210> 6 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR27a-3p <400> 6 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatc actcagtgac 60 tggagttgct tggctctggt gtattggtgc ggaacttagc cactgtgaa 109 <210> 7 <211> 109 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR31-5p <400> 7 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagatt acagcgtgac 60 tggagttgct tggctctggt gtattggtag ctatgccagc atcttgcct 109 <210> 8 <211> 110 <212> DNA <213> Artificial Sequence <220> <223> Sensor DNA for detecting hsa-miR92a-3p <400> 8 aacaatacca cgaccaccga caactacacg ctacagtcgc atacgagata agtcagtgac 60 tggagttgct tggctctggt gtattggtac aggccgggac aagtgcaata 110 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR486-5p <400> 9 cgctacagtc gcatacgaga tttaggc 27 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR486-5p <400> 10 tcctgtactg agctgccccg ag 22 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR210-3p <400> 11 ctacagtcgc atacgagata catcg 25 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR210-3p <400> 12 ctgtgcgtgt gacagcggct ga 22 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR1290 <400> 13 gctacagtcg catacgagat tcattc 26 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR1290 <400> 14 tggatttttg gatcagggaa ccaa 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR132-3p <400> 15 gcatacgaga tggcaacgtg 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR132-3p <400> 16 taacagtcta cagccatggt cg 22 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR146a-5p <400> 17 cagtcgcata cgagattagc ttg 23 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR146a-5p <400> 18 tgagaactga attccatggg ttac 24 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR27a-3p <400> 19 gcatacgaga tcactcagtg ac 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR27a-3p <400> 20 ttcacagtgg ctaagttccg c 21 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR31-5p <400> 21 cagtcgcata cgagattaca gc 22 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR31-5p <400> 22 aggcaagatg ctggcatagc t 21 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for detecting hsa-miR92a-3p <400> 23 tcgcatacga gataagtcag tg 22 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for detecting hsa-miR92a-3p <400> 24 tattgcactt gtcccggcct 20
Claims (10)
b) 상기 센서 DNA의 모듈 영역을 주형으로 하고, 상기 타겟 RNA를 프라이머로 하여 중합효소로 중합하는 단계; 및
c) 실리카를 처리하여 타겟 RNA와 혼성화되지 않은 센서 DNA를 제거하는 단계를 포함하는, RNA 검출 방법.a) hybridizing the sensor DNA including the complementary sequence of the target RNA to be detected with the target RNA;
b) using the module region of the sensor DNA as a template and polymerizing the target RNA as a primer with a polymerase; and
c) treating the silica to remove the sensor DNA that is not hybridized with the target RNA.
상기 b) 단계의 중합 단계를 통해 형성된 중합된 가닥을 c) 단계 이후PCR 반응을 통해 증폭하는 단계를 더욱 포함하는, RNA 검출 방법.According to claim 1,
Further comprising the step of amplifying the polymerized strand formed through the polymerization step of step b) through a PCR reaction after step c), RNA detection method.
상기 타겟 RNA는 스몰(small) RNA인 것인, RNA 검출 방법.According to claim 1,
The target RNA is a small (small) RNA, RNA detection method.
상기 실리카는 pH 7.5 내지 pH 8.5의 조건에서 처리되는 것인, RNA 검출 방법.According to claim 1,
Wherein the silica is treated under the conditions of pH 7.5 to pH 8.5, RNA detection method.
상기 센서 DNA는 타겟 RNA를 인지하여 혼성화하고,
상기 실리카는 타겟 RNA와 혼성화되지 않은 센서 DNA를 제거하는 것인, RNA 검출용 조성물.7. The method of claim 6,
The sensor DNA recognizes and hybridizes to the target RNA,
The silica is to remove the sensor DNA that is not hybridized with the target RNA, RNA detection composition.
상기 센서 DNA는 단일 가닥(single strand, ss) DNA 형태인 것인, RNA 검출용 조성물.7. The method of claim 6,
The sensor DNA is a single strand (single strand, ss) DNA in the form of, RNA detection composition.
상기 실리카는 pH 7.5 내지 pH 8.5의 조건에서 처리되는 것인, 단일가닥 DNA(single strand DNA, ssDNA) 제거 방법.10. The method of claim 9,
The silica is treated under the conditions of pH 7.5 to pH 8.5, single-stranded DNA (single strand DNA, ssDNA) removal method.
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