This work was supported, in whole or in part, by NIH Grants R01 AA 020758-04, U01DK 061731-13 and T32 DK 007150-38 to AJS and T32 DK 007150-38 to AA. This is original work and is not under consideration elsewhere for publication.

혈액 샘플 1. 컬렉션 <ol><li> 전주 포사에서 눈에 띄는 정맥의 표준 정맥 천자에 의해 (다른 항응고제에 비해 몇 가지 장점이 있습니다) 항응고제 에틸렌 디아민 테트라 아세트산 (EDTA)를 포함하는 10 ML의 플라스틱 튜브에 말초 정맥 혈액 샘플을 금식 수집합니다. </li><li> 적혈구 및 RNA 소량의 자유 혈장을 얻었다 탁상 원심 분리기로 4 ℃에서 20 분 동안 1,600 XG에서 혈액 샘플을 원심 분리기. </li><li> 순차적으로 각각 남아있는 세포 파편을 제거하기 위해 WBC 및 혈소판 15 분 후 추가를 제거하는 10 분 동안 스윙 버킷 로터에서 3,000g (4 °에 C)에서 상층 액을 원심 분리. </li><li> 제조 지시에 따라 RT에서 농도계를 이용하여 플라즈마의 밀도를 측정한다. 주 : 농도 조정 (d = 1.023 g / ㎖) 0.9 % 식염수 용액 엑소 좀을 제거하지만, 종래 밀도 구배 초 원심 분리 후 필요하다. </li></올&gt; 플라즈마 2. 엑소 좀 제거 <ol><li> HDL 유사한 밀도가 순환 엑소 좀을 제거하고 miRNA의 (3)의 양적 중요한 소스를 나타냅니다. <ol><li> 4 ° C에서 30 분 동안 배양 한 다음 1 ml의 플라즈마에 252 ㎕의 엑소 좀 침전 솔루션을 추가하여이 작업을 수행합니다. 엑소 좀을 펠릿, 4 ℃에서 1,500g에서 30 분 동안 혼합물을 원심 분리. </li><li> HDL, 밀도 구배 초 원심 분리하여 더 처리 카보네이트 두꺼운 초 원심 분리 튜브에 얻어진 상등액 전송 한 용액을 분리 (아래 참조). </li></ol></li></ol> 3. 밀도 구배 초 원심 분리 (그림 1). <ol><li> HDL은 고정 각 로터 448,811 X G에서 동작하는 8 ° C 각각과 바닥 초 원심 분리기를 사용하는 3 단계 프로세스를 사용하여 분리해서한다. </li><li> 세 가지 다른 밀도 솔루션 순차적으로 각각의 분리를위한 신선한를 준비합니다. <ol><li> 용액 A를 준비 (VLDL의 분리 D = 1.006 g / ㎖) 11.4 g의 NaCl (염화나트륨 : 0.195 몰)을 용해하여 멸균 증류수 1,000 ㎖에 0.1 g의 EDTA2Na 1 ml의 1N NaOH를합니다. 그런 다음 멸균 증류수의 추가 3 ㎖를 추가합니다. </li><li> 100 ㎖ 용액 A (염화나트륨 0.195 몰, NaBr은 2.44 몰)을 25.2 g의 NaBr은을 추가하여 용액 B를 준비 (LDL의 격리, D = 1.182 g / ㎖). </li><li> 용액 A의 100 ㎖ (염화나트륨 0.195 몰, NaBr은 7.7 몰) 78.8 g의 NaBr은을 혼합하여 (D = 1.470 g / ㎖ HDL의 분리) 솔루션 C를 준비합니다. 농도계를 사용하여 실온에서 적절한 밀도를 확인합니다. 더 사용할 때까지 4 ° C에서 모든 솔루션을 보관하십시오. </li></ol></li></ol> VLDL 4. 분리 <ol><li> 플라즈마 1 ㎖ (평균 밀도 = 1.023 g / ㎖) 6.5 ㎖의 폴리 카보네이트 두꺼운 벽으로 둘러싸인 초원 심 분리기 튜브에 지방 레드 7B와 뉴 클레아 무료 200 μl를 섞는다. </li><li> 조심스럽게 혼합 위에 A 액 5 ㎖ 계층. 필요한 경우, 용액 A의 t의 상단에 추가로 지방 레드 7B를 추가O 각 튜브의 무게 균형. - 2 시간 (5 가속도)에 대한 원심 분리기 (감속 - 7). 주 : 원심 동안 지단백질가 평형 농도 영역에서 대역으로 축적된다. </li><li> 실행의 끝에서 2 층을 관찰합니다. 4 ° C에서 상단 층 및 저장소를 나타내는 VLDL 분획의 1.5 ml의를 제거합니다. </li><li> 마지막으로, LDL을 포함하는 튜브의 바닥으로부터 피펫 전사 4 용액을 이용하여, HDL, LDL을 분리하기위한 새로운 폴리 카보네이트 튜브 알부민 및 지방산 분획. </li></ol> LDL 5. 분리 <ol><li> 각각 LDL과 HDL 비율 (4 절)를 포함하는 튜브에 용액 B 100 ㎕를 뉴 클레아 무료 지방 레드 7B 2 ㎖를 섞는다. </li><li> 그런 다음 3 시간 (가속 9, 감속 7)을 위해 밖으로 원심 분리기. 그 후, 상단 층을 나타내는 LDL 분획의 1.5 ml의 제거 및 -80 ° C에서 4 °의 C 또는 저장하시오. 마지막으로, HDL을 함유하는 튜브의 바닥에서 4 ml의 전송새로운 폴리 카보네이트 튜브 부분. </li></ol> HDL 6. 분리 <ol><li> 이 용액 C의 용액, 100 μL 클레아없는 지방 레드 7B 및 각각 HDL 분획을 함유하는 튜브에 98 % β 메르 캅토 에탄올의 15 μl를 섞는다. </li><li> 3 시간 (가속 9, 감속 7)에 대한 원심 분리기. 그 후, 상부 층을 나타내는 HDL 분획이 용액을 제거하고 -80 ℃에서 4 ℃에서 보관하거나 저장하거나. </li></ol> 7. 탈염 및 지단백질 분획의 농도 <ol><li> 이후의 아가로 오스 겔 전기 영동 및 PCR과의 간섭을 방지하기 위해, 제조업체의 지침에 의해 설명 된 바와 같이 (LDL / HDL에 대한 VLDL 및 10K 튜브에 대한 3K 관) 적절한 분자량의 컷오프와 원심 필터 장치를 사용하여 밀도 구배 초 원심 분리 동안 추가 과도한 소금을 제거합니다. <ol><li> 간단히, 차가운 PBS (137 mM의 염화나트륨, 2.7 mM의 KCL, 8 밀리미터의 Na2HPO4, 2 mM의 KH2PO4, pH를 7.4) 2.5 ml에 추가 한 후 centrifuGE 전체 VLDL 분획 수집-동안 밀도 구배 초 원심 분리 4 ° C에서의 스윙 버킷 회 전자를 사용하여 60 분 동안. </li><li> 30 분마다 10 ml의 빙냉 PBS로 두 번 LDL 분획을 탈염. 다음으로, HDL 분획을 탈염 두 번 13 ml의 얼음 차가운 PBS를 사용합니다. 높은 PBS 부피는 아가 로스 겔 전기 영동으로 이동도를 개선하는 것이 필요하다. 원심 분리 후, 지질 단백질 함유 용질을 제거하고 4 ° C에서 보관 또는 -80 ° C에 저장됩니다. </li></ol></li></ol> 8. 아가 로스 젤 전기 영동 <ol><li> 다음과 같이 제조업체의 지침의 약간의 수정과 함께 키트를 사용 지단백 아가로 오스 겔 전기 영동을 Perfrom. 참고 :이 단계는 집중 지질 단백질 샘플의 품질과 순도를 평가하기 위해 단지이다. <ol><li> 요약하면, 밀도 구배 초 원심 분리하여 탈염 지단백질 분획 6 μl를 구해서 미리 주조 지단백질 젤 상에 로딩. 인간의 lipop를 사용하여크기 기준으로 VLDL, LDL과 HDL에 대한 rotein 표준. 담당자 준비 버퍼를 사용하여 60 분 동안 100 V에서 RT에서 전기 영동을 수행하십시오. </li><li> 10 분 동안 겔을 건조하고 지방 레드 7B와 함께 RT에서 10 분 동안 염색. 5 분 동안 다시 메탄올 - 물 75:25 (v / v)의 건조 혼합물에 겔 Destain. </li></ol></li></ol> 9. RNA 추출 및 정제 <ol><li> 혈청 / 플라즈마 miRNA의 분리 및 정제 키트를 사용하여 정제 된 인간의 HDL에서의 miRNA의 분리를 Perfrom. <ol><li> 간단히하는 vortexer를 혼합 한 후 핵 단백질 복합체 및 RNases의 불 활성화의 완전한 분리를 보장하기 위해 RT에서 5 분 동안 배양, 정제 HDL 200 μL에 RNA 용해 시약 1 ㎖를 추가합니다. </li><li> (1.6 × 10 8 복사 / μL CEL-은 miR-39) 혼합물에 다음 합성 Caenorhabditis 엘레 간스 마이크로 RNA의 3.5 μl를 스파이크. 그런 다음, 제조자의 지시에 따라 RNA의 추출을 실시한다. </li></ol></li><li> purificati를 수행제조업체의 지침에 따라 용출 스핀 열 추출-miRNA의의에. spectrophorometer와 정제 HDL에서의 miRNA의 농도를 측정한다. 참고 : 스핀 컬럼에서의 miRNA의 용출 된 RNase없는 물 16 μl를 고용했다. </li></ol> 10. 역전사 (RT-PCR) <ol><li> HDL에서의 miRNA 100 NG를 분리하여 miRNA의 합성 (CEL-은 miR-39)과 역전사 키트를 이용하여 제조자의 지시에 따라 20 ㎕의 반응 부피로 역전사 타서. </li><li> 템플릿의 miRNA (NTC)없이 역전사 효소 믹스 (NRT)없이 적절한 제어를 수행합니다. </li></ol> 11. 실시간 PCR (QRT-PCR) <ol><li> cDNA를 2 희석, 10 ㎕의 PCR 혼합물, 2 μL 유니버설 프라이머 2의 miRNA 프라이머 μL, 4 ㎕의 RNA 분해 효소가없는 물 : 1 2 ㎕를 20 ㎕의 총 부피로 리얼 타임 PCR을 Perfrom. </li><li> 를 Reacti을 실행15 초, 30 초 동안 55 ° C 및 30 ~ 70 ° C의 확장 단계에 94 ° C의 45주기 다음 15 분 동안 95 ° C에서 96 웰 플레이트에서의 삼중의 모든 반응을 Perfrom s.ec . </li><li> 다음으로, 제조업체의 지침에 따라 합성 하우스 키핑 유전자에 정상화 후 2 -ΔΔ 코네티컷 방법을 사용하여 miRNA의의 Calcuate 상대적인 양. </li></ol>

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저자는 공개 아무것도 없어.

혈액에서 새로운 바이오 마커의 식별은 각종 질병의 임상 진단 및 예후에 도움이됩니다. 마이크로 RNA는 바이오 마커의 모든 자질을 가지고하는 것으로 알려져하고 다양한 연구 14-17에 표시되어있다. 이 연구에서 우리는 플라즈마 HDL에서의 miRNA를 분리하기 위해 신속하고 간단한 쉬운 방법을 증명하고있다. VLDL, LDL 및 HDL의 분리의 종래의 밀도 구배 초 원심 분리 방법은, 플라즈마의 정확한...

MicroRNAs are endogenous non-coding tiny RNA species that are highly conserved and are considered key players in the regulation of various biological processes by degrading or repressing specific target messenger RNAs1. Because miRNAs act intracellularly they have been explored as tissue-derived biomarkers which led to the discovery of tissue-specific functions of these miRNA. However, miRNAs are also found extracellularly either associated with proteins or in exosomes/micro vesicles that effectively can shield them from degradation by extracellular RNases2. More recent studies have shown that the protective effect of HDL may not be closely linked to its capability to promote cholesterol efflux but rather to its non-cholesterol cargo, in particularly as a circulating miRNAs carrier 3, 4. These miRNAs may not only modulate lipid metabolism but are also associated with anti-inflammatory, antioxidant and antithrombotic effects of the HDL-miRNA complex 5, 6.
To further explore the role of miRNAs carried in HDL particles, a simple and easy protocol needs to be established for miRNA extraction from isolated highly purified HDL for use in clinical routine. Numerous methods have been described to isolate HDL. These methods are either very time consuming or require large volume of plasma that may require sample pooling, extensive dialysis for desalting isolated lipoproteins and they do not completely remove exosomes as a source of miRNAs3, respectively. Here we describe a simple and rapid method that can isolate miRNA from highly purified HDL utilizing small volume of blood samples on a larger scale. We believe that this method may serve as good reference to promote research into the role of circulating miRNAs and in particular the role of HDL in facilitating communication between various cells and organs.

<table><tbody><tr><td>Plastic Vacutainer Lavender K2EDTA tubes&amp;nbsp;</td><td>Becton, Dickinson and Company</td><td>366643</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific, Sorvall Legend X1R&amp;nbsp;</td><td>75004261</td><td></td></tr><tr><td>Densito 30PX densitometer</td><td>Mettler Toledo</td><td>MT51324450</td><td></td></tr><tr><td>ExoQuick solution&amp;nbsp;</td><td>Invitrogen</td><td>4484451</td><td></td></tr><tr><td>Polycarbonate thick-walled ultracentrifuge tube</td><td>Thermo Scientific</td><td>O3237</td><td></td></tr><tr><td>Sorvall WX100 ultracentrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td>46902</td><td></td></tr><tr><td>Fat Red 7B&amp;nbsp;</td><td>Sigma-Aldrich</td><td>201618</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal filter devices 10K</td><td>Millipore</td><td>UFC901008</td><td></td></tr><tr><td>Amicon Ultra-centrifugal filter devices 3K</td><td>Millipore</td><td>UFC800308</td><td></td></tr><tr><td>QuickGel Lipo kit&amp;nbsp;</td><td>Helena Laboratories&amp;nbsp;</td><td>3344,3544T</td><td></td></tr><tr><td>Human lipoprotein standards for VLDL, LDL and HDL</td><td>LipoTrol; Helena Laboratories</td><td>5069</td><td></td></tr><tr><td>Rep Prep buffer&amp;nbsp;</td><td>Helena Laboratories&amp;nbsp;</td><td>3100</td><td></td></tr><tr><td>RNeasy MinElute spin columns&amp;nbsp;</td><td>Qiagen</td><td></td><td></td></tr><tr><td>NanoDrop 1000 analyzer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>miScript II RT Kit&amp;nbsp;</td><td>Qiagen</td><td>218161</td><td></td></tr><tr><td>CFX96 Touch real-time PCR detection system</td><td>BioRad</td><td></td><td></td></tr><tr><td>miRNeasy Serum/Plasma Kit</td><td>QIAGEN</td><td>217184</td><td></td></tr><tr><td>miScript Primer Assays</td><td>QIAGEN</td><td>141078139</td><td></td></tr><tr><td>miScript SYBR Green PCR Kit&amp;nbsp;</td><td>QIAGEN</td><td>218073</td><td></td></tr><tr><td>miRNeasy Serum/Plasma Spike-In Control</td><td>QIAGEN</td><td>219610</td><td></td></tr><tr><td>NaOH</td><td>SIGMA-ALDRICH</td><td>480878</td><td></td></tr><tr><td>0.20 &amp;#181;M sterile syringe filter</td><td>SIGMA-ALDRICH</td><td>Z227536</td><td></td></tr></tbody></table>

fast and simplified method for high through-put isolation of mirna from highly purified high density lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

엑소 좀 제거 후 고밀도 지단백의 분리 고순도 HDL에서의 miRNA를 얻으려면이 miRNA의 오염 (7)의 소스를 대표하는 엑소 좀을 제거 할 필요가있다. 이것은 상업적으로 이용 가능한 키트 밀도 구배 초 원심 분리에 앞서 수행 하였다. 실용적으로 상업 회사에 의해 개발 된 3 단의 ​​표준 밀도 구배 초 원심 프로토콜 (도 1)를 변...

Watch this Scientific Journal Video about Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein at JoVE.com

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

마이크로 RNA는 중요한 조절 역할을하고 다양한 인간 질병에 대한 새로운 치료 대상으로 떠오르고있다. miRNAs의 고밀도 지단백질에 수행되는 것을 도시하고있다. 우리는 빠른 속도로 인간 혈장에서 miRNA의 분석에 적합한 정제 HDL을 분리하는 간단한 방법을 개발했다.

고도로 정제 된 고밀도 지단백질에서의 miRNA의 높은 넣어 관통 분리에 대한 신속하고 단순화 된 방법

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

fast-simplified-method-for-high-through-put-isolation-mirna-from

Research

JoVE Journal

Biology

11.9K 조회수.  Virginia Commonwealth University.  마이크로 RNA는 중요한 조절 역할을하고 다양한 인간 질병에 대한 새로운 치료 대상으로 떠오르고있다. miRNAs의 고밀도 지단백질에 수행되는 것을 도시하고있다. 우리는 빠른 속도로 인간 혈장에서 miRNA의 분석에 적합한 정제 HDL을 분리하는 간단한 방법을 개발했다. 

비디오: Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

작은 RNA 정량 비는 코딩을위한 고밀도 지방 단백질의 분리

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

연구

생화학

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

높은 처리량 MicroRNA의 프로파일 : 최적 멀티 플렉스 플루 동적 ArrayTM의 IFCs에 Nanoliter의 규모 qRT - PCR

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

생체공학

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

마이크로 로르 나를 사용한 네이티브 단백 입자의 농축과 그에 따른 절대/상대 미세 유 변 함량 및 셀룰러 전송 속도의 후속 측정

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.

극심분리에 의한 혈장 지단백질의 격리 및 분석

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

격리 및 프로파일 링 마이크로 RNA-포함하는 인간의 담즙에서 엑소 좀을

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

의학

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

혈액과 문화 매체에서 파생 Exosomes의 정화 및 마이크로 RNA 프로파일

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

생물학

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

인간 혈청에서 작은 비 암호화 RNA를의 분리

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

높은 처리량 시퀀싱에 의한 마이크로 RNA 정량을위한 작은 RNA 분자의 고효율 내고

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

음 세포 기관이 격리 키트를 사용 하 여 급속 한 지질 작은 물방울 격리 프로토콜

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

Circulating miRNA Extraction: A Method to Isolate miRNA from Plasma Samples Via Organic Extraction and Small RNA Enrichment

Source: Canale, M. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/58615"> Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer.</a> J. Vis. Exp. (2019).
This video describes the extraction of circulating microRNA from plasma samples of colorectal cancer patients using organic extraction followed by glass-fiber column enrichment. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis, and treatment decision-making and monitoring. This method will help identify new circulating biomarkers capable of predicting clinical outcomes in patients.

순환 miRNA 추출: 유기 추출 및 Small RNA 농축을 통해 혈장 샘플에서 miRNA를 분리하는 방법

Source: Canale, M. et al.,  Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer. J. Vis. Exp. (2019).
This ...