작품은 약물 남용 센터 국립 연구소 (P50 DA 011806)와 국방의 미국 육군과 (W81XWH - 07 - 1-0183)에 의해 부분적으로 지원되었다.

1. 사용자 정의 miRNA의 microarrays의 인쇄 <ol><li> microarray는 대학이나 회사에서 microarray 핵심 시설 서비스를 이용 슬라이드 인쇄합니다. microarray 제조의 품질은 microarray 실험의 성공을 위해 가장 중요한 요소 중 하나입니다. 가능하면 몇 가지 서비스를보십시오. 이상적으로, 하나는 한 번에 50-100 슬라이드를 인쇄 것이며, 모든 슬라이드가 잘 구분하여, 각각의 장소와 동일 보입니다. </li><li> microarray 지원, 우리는 격차 II 코팅 슬라이드를 사용합니다. </li><li> miRNA 프로브 위해, 우리는 NCode 멀티 종 (species)이 Microarray 프로브가 V2 세트 miRNA 사용합니다. </li><li> 10 μm의 3 X SSC에있는 모든 oligonucleotides를 해산하고 quadruply 슬라이드 그들을 인쇄할 수 있습니다. GAAPS II 슬라이드에 대한 지침에 따라 자외선 조사에 의해 슬라이드를 수정. 인쇄 프로브와 함께 지역과 측면을 표시하는 다이아몬드 펜으로 슬라이드 레이블. 22 스토어 ° C. </li></ol> 2. 샘플 준비 <ol><li> 모든 방법또는 시약이 한 그것이 작은 RNAs를 유지로 RNA를 분리하는 데 사용할 수 있습니다. 우리는 일반적으로 260nm와 280nm 판독하여 측정 RNA의 준비 만족 수량 및 품질과 함께, 총 RNA를 추출하기 위해 Trizol (Invitrogen)을 사용합니다. </li><li> RNA 라벨, 1 X 버퍼 (50 MM의 HEPES, pH를 7.8, 20 MM MgCl 2, 10 μg / ML BSA, 10 % 5&#39; - pCU - DY547 - 3 &#39;6 0.5 μg과 함께 총 RNA의 믹스 ~ 25 μg을 위해 DMSO) 포함 ~ 20 대 T4 RNA ~ 10 밀리미터 DTT와 ATP에 대한 10 X T4 RNA Ligase 한 버퍼의 ~ 0.02-0.03 최종 볼륨 보충 Ligase 1. </li><li> 2-24시간의 냉장고 안에 얼음 진행 라벨 수 있습니다. 0.3 M의 아세트산 나트륨과 에탄올 3 권와 RNA를 침전. 효율적인 결합과 강수량 들어, 이상이 MG / ML에서 총 RNA를 해산하고, ~ 20 μl의 총 내고 볼륨을 유지. <ol><li> 적은 RNA를 사용할 경우, 입력 RNA 및 5&#39;가 - pCU - DY547 - 3 &#39;는 크기를 줄일 수 있습니다의 금액입니다. &lt;/ 리&gt; <li> RNA가 매우 묽은 경우, 강수량을 도울 효모 tRNA 등 통신 사업자를 추가합니다. </li></ol></li><li> Ulysis 알렉사 형석 하이브 리다이 제이션 프로세스 제어로 647 핵산 라벨링 키트 6을 사용 포유 동물 miRNAs의 하위 집합에 해당 참조의 DNA oligonucleotides 총 1 μg의 결합 및 레이블. CentriSep 칼럼을 사용하여 표시된 DNA를 정화하고 -20 ° C.에 어두운에 저장 </li></ol> 3. Microarray의 하이브 리다이 제이션 <ol><li> 처음으로 슬라이드를 사용하려면, 37 3 X SSC, 0.1 % SDS, 30-60 분 0.2 % BSA의 필터링 솔루션에 슬라이드를 미리 잡종 ° C. 잠수함 물이 몇 번에서 다음 이소프로판올에. 22 5 분 100g에서 슬라이드 어댑터 ° C.있는 원심 분리기를 사용하여 슬라이드를 건조 </li></ol> 참고 : 우리는 microarray의 하이브 리다이 제이션을 수행하기 위해 코닝 microarray의 하이브 리다이 제이션 챔버와 Lifterslips의 에리 과학의 mSeries을 사용합니다. <ol start="2"><li> 리 씻어공기 건조하기 전에 물에 fterslips 다음 에탄올로. microarray의 하이브 리다이 제이션 챔버 기지 내부에 슬라이드와 슬라이드의 위에 lifterslip 놓으십시오. lifterslip은 슬라이드 연락은 흰색 스트립과 탐사 영역을 커버합니다. </li><li> 어둠 속에서, 레이블 RNA를 시켰던을 돌린다. 70 % 에탄올로 한 번 씻고, 그리고 공기는 펠렛을 건조. 펠렛은 붉은해야합니다. </li><li> 400 MM 나 2 HPO 4 산도 7.0, 0.8 % BSA 5 % SDS, 정화의 1/15-1/100 μl와 12 % 포름 아미드 6, RNA 샘플마다 라벨 참조 DNA (2.4)이있는 하이브 리다이 제이션 솔루션을 준비합니다. 추가 참조 DNA의 양은 다른 oligonucleotides는 (2.4) 얼마나 많은 레이블 아르에 따라 달라집니다. 100 개 이상있다면 덜 희석이 필요합니다. </li><li> 하이브 리다이 제이션 솔루션 ~ 60 μl로 잘 RNA 펠렛를 해산. </li><li> 코닝 microarray의 하이브 리다이 제이션 챔버 자료의 가습 우물의 물을 각각 20 μl를 추가합니다. </li><li> t 추가슬라이드에 표시 RNA 및 참고 DNA의 그 혼합물. 얇은 pipet 팁을 사용하여 부드럽게 lifterslip의 가장자리를 터치하고, 솔루션은 흡입을 통해 lifterslip 및 슬라이드 사이의 공간을 입력할 수 있습니다. </li><li> 하이브 리다이 제이션 챔버가베이스 커버를 통해 장소, 그때 금속 클립과 챔버를 봉인. </li><li> 코닝에서 챔버 카세트와 함께 제공되는 비닐 봉투 안에 전체 챔버 놔. </li><li> 침수 종이 타월로 용기 내부 챔버 카세트 (들)을 장소, 플라스틱 포장과 용기를 커버하고 37 안에 자리 ° ~ 24 시간 동안 C 습기, 휴대 문화 인큐베이터. 이러한주의 사항 (3.6, 3.9, 3.10)은 하이브 리다이 제이션 솔루션이 부화하는 동안 밖으로 건조하지 않도록. </li></ol> 4. 포스트 하이브 리다이 제이션 처리 <ol><li> 챔버 카세트를 하나씩 분해. 22 2 X SSC에 잠수함 슬라이드와 lifterslip ° C. lifterslip 자연 슬라이드 떨어질 것입니다. 0.8 X SSC에 놓으십시오. 빨래두 번, 그리고 0.8 X SSC에있는 슬라이드 0.4 X SSC, 총 1-2 분 안에 세 번. 슬라이드 어댑터와 원심 분리기를 사용하여 슬라이드를 건조시킵니다. 물로 lifterslips을 씻고 나중에 다시 사용하기 위해 저장할 수 있습니다. </li><li> 모든 적합한 스캐너, 예를 들어, Perkin 엘머 ScanArray 5000 또는 분자 장치 축삭 GenePix 4000B microarray 스캐너와 슬라이드를 검사합니다. 해당 이미지 파일을 얻기 위해 파장 547 nm의 가까이에와 647 nm의에서 스캔. </li><li> 입력, 4.2에서 이미지 파일)에서 픽셀 농도를 정할 같은 BlueFuse 같은 프로그램을 사용합니다. 추가 고려 대상에서 microarrays에 대한 비정상적인 명소를 제외하는 프로그램을 각각의 장소를 검사합니다. 이러한 비정상적인 명소는 일반적으로 가난한 슬라이드 인쇄하거나 슬라이드 하이브 리다이 제이션 후 처리에서 발생. </li><li> 데이터 분석 및 프레 젠 테이션 등 GeneSpring 및 Excel과 같은 프로그램을 사용합니다. microarray 데이터 정상화를위한 일반적인 고려 사항은이 문서의 범위를 넘어있는, 적용됩니다. </li><li> 일단 만족스러운 신호는 AF를 얻을 수 있습니다ter 4.3), 재사용 7 microarray 슬라이드를 스트립. 10~20분를위한 62의 얼룩 접시에 미리 예열, 1 ㎜ NaOH 및 0.1 X SSC ° C에서 다음 젖어, 물속에 슬라이드를 씻어. 일단 부화를 반복합니다. 22까지 부드럽게 흔들어 60 분 동안 물에 몇 번 슬라이드 ° C. 씻으십시오 원심 분리기를 사용하여 슬라이드, 22에서 저장 ° C. 건조 </li><li> 재생 슬라이드를 사용하려면, 다시 그들을 미리 잡종 필요가 없습니다. 간단히 이소프로판올 다음 물 속에 그들을 씻고, 바로 하이브 리다이 제이션 전에 슬라이드를 건조. </li></ol> 5. 대표 결과 : 우리는 대부분 샘플 수천 글로벌 miRNA 발현을 프로필에 설명된 절차를 따라했습니다 즉, 여러 조건 하에서 인간의 표본으로 zebrafish에서 격리 RNAs가. 그림 1은 매우 정확하고 강력한 하이브 리다이 제이션 신호를 증명하기 위해 microarray의 스캔 이미지를 보여줍니다 슬라이드. 피어슨 correlati기술 사이의 계수에 대한 microarray의 하이브 리다이 제이션의 복제 우수한 재현성을 나타내는 ~ 0.99 7, 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3250/3250fig1.jpg" /> 하이브 리다이 제이션 후 스캔 miRNA의 microarray 슬라이드 1 복합 이미지를 그림. 노란색 반점이 동일한 프로브에 DNA와 RNA 모두에 의해 hybridizations에서 동안 레드 명소 참조 DNA, DY547 - 라벨 RNA 샘플에서 녹색 관광 명소로 hybridizations에서 결과.

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관심 없음 충돌 선언하지 않습니다.

깊은 시퀀싱 기술의 최근 발전에도 불구하고, microarray는 DNA와 RNA의 높은 처리량 분석을위한 가능한 선택 남아있다. 깊은 시퀀싱에 비해 microarray 실험은 저렴하고 있으며, 전형적인 분자 생물학 연구소의 실험과 유연성을 허용하고 시간을 절약할 수 데이터 분석 사내의 대부분을 수행할 수 있습니다. 앞으로, microarrays 가능성이 적합 집중적으로 유전자, 예를 들어 세트를 심문하러 있으며, 전부 또?...

<table border="1"><tbody><tr><th> 항목 </th><th> 공급 업체 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> NCode 멀티 종 Microarray 프로브 세트 V2를 miRNA </td><td> Invitrogen </td><td> MIRMPS201 </td><td> miRBase 릴리스 9.0 (2006 년 10 월)에 따라 설계되었습니다. 그것은 1140 ~ 수정되지 않은, 웜, 비행, zebrafish, 마우스, 쥐, 인간 miRNAs을 위해 프로브로 34-44 NT 긴 oligonucleotides, 그리고 snoRNAs 등 내부 통제 프로브의 숫자가 포함되어 있습니다. miRNA 프로브는 따라서 성숙 miRNAs ~ 44 NT의 크기 보완 시퀀스 doublets 있습니다. 분석을 위해 하나가 관심의 특정 게놈 (S)에서 miRNAs에 초점을 맞출 수 있습니다. </td></tr><tr><td> Trizol </td><td> Invitrogen </td><td> 15596018 </td><td> 총 RNA 샘플 빠르고 준비하고 수치 쉽고 같은 mRNA 분석과 같은 다운 스트림 애플 리케이션에 적합하지만 우리는 또한 사용, 라벨에 대한 작은 RNA 분획을 농축 있습니다. </td></tr><tr><td> T4 RNA Ligase 일 </td><td> 뉴 잉글랜드 Biol복근 </td><td> M0204L </td><td></td></tr><tr><td> Ulysis 알렉사 형석 647 핵산 라벨링 키트 </td><td> Invitrogen </td><td> U21660 </td><td> 이 키트 또는 유사한 제품뿐만 아니라 실험적인 RNA 샘플 또는 컨트롤 RNA를 (대신 제어 DNA의) 라벨을 사용하실 수 있습니다. </td></tr><tr><td> 5&#39; - pCU - DY547 - 3 &#39; </td><td> Dharmacon </td><td> 사용자가 만든 </td><td> 작은 RNA 분획은 비슷하게 내고으로 분류하실 수 있습니다. </td></tr><tr><td> CentriSep 열 </td><td> 프린스턴 분판 </td><td> CS - 901 </td><td></td></tr><tr><td> GAPSII 코팅 슬라이드 </td><td> 코닝 </td><td> 40,004 </td><td> 슬라이드의 다른 유형도 사용할 수 있습니다. </td></tr><tr><td> Microarray의 하이브 리다이 제이션 챔버 </td><td> 코닝 </td><td> 2551 또는 40080 </td><td> 하이브 리다이 제이션 실 및 coverslips 다른 종류도 작동합니다. 상용 하이브 리다이 제이션 기계를 사용하면 ~ 2 시간, 크게 예 하이브 리다이 제이션 시간을 줄일 수 있습니다. </td></tr><tr><td> Lifterslips </td><td> 일ermo / 에리 과학 </td><td> 25X60I - M5439 - 001 - LS </td><td></td></tr><tr><td> BlueFuse </td><td> BlueGenome </td><td></td><td></td></tr><tr><td> GeneSpring </td><td> 애질런트 </td><td></td><td></td></tr></tbody></table>

performing custom microrna microarray experiments

microRNAs는 (miRNAs) 널리 eukaryotes 1 표현됩니다 ~ 22 세포핵 (NT) 긴 RNA 분자의 큰 가족입니다. 복잡한 genomes는 주로 사후 transcriptionally 대상 유전자 2, 3의 방대한 숫자의 표현을 억제 miRNAs 중 적어도 수백, 인코딩. miRNAs는 생물 학적 과정 1 광범위한 제어합니다. 또한, 변경 miRNA 발현이 같은 암 등 인간의 질병와 관련된되었으며, miRNAs는 질병 및 예후 (豫后) 4, 5에 대한 생체 역할을 수 있습니다. 그것은 표현과 여러 가지 조건에서 miRNAs의 기능을 이해하기 위해, 따라서, 중요합니다. 실시간 PCR, microarray, 깊이 시퀀싱 : 세 가지 주요 방법은 프로필 miRNA 발현에 고용되었습니다. miRNA의 microarray의 기술은 일반적으로 저렴 높은 처리량되는 장점을 가지고 있으며, 실험 및 분석 단계의 대부분은 카 수 있습니다대부분의 대학, 의과 대학 및 관련 병원에서 분자 생물학 연구실에서 지휘를 맡는​​. 여기, 우리는 사용자 정의 miRNA의 microarray 실험을 수행하는 방법을 설명합니다. miRNA 프로브 세트는 miRNA의 microarrays를 만들어 유리 슬라이드에 인쇄됩니다. RNA는 작은 RNA 수종을 보존하는 방법이나 시약을 사용하여 절연하고 형광 염료와 함께 표시됩니다. 컨트롤로서, miRNAs의 하위 집합에 해당 참조의 DNA oligonucleotides는 다른 형광 염료로 분류됩니다. 참조 DNA는 슬라이드와 하이브 리다이 제이션의 품질을 증명하기 위해 제공되며, 또한 데이터 정규화에 사용됩니다. RNA와 DNA가 혼합하여 데이터베이스에있는 miRNAs 대부분의 프로브를 포함하는 microarray 슬라이드에 hybridized입니다. 세탁 후, 슬라이드 이미지를 얻기 위해 스캔하고, 개별 명소의 농도는 계량입니다. 이러한 원시 신호는 추가로 처리하고 해당 miRNAs의 표현 데이터로 분석됩니다. Microarray 슬라이드는 옷을 벗긴 채 microarrays의 비용을 절감하고 microarray 실험의 일관성을 향상시키기 위해 생성하실 수 있습니다. 동일한 원칙과 절차를 정의 microarray 실험의 다른 유형에 적용됩니다.

Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

Performing Custom MicroRNA Microarray Experiments

사용자 정의 microRNA의 microarray 실험을 수행 간단한 절차 설명합니다. 단계, RNA, RNA 라벨링 및 참조 DNA를 분리 microarrays에 샘플을 hybridizing, microarrays를 검색, quantifying 및 하이브리드화 신호를 분석 등이 있습니다.

사용자 정의 MicroRNA의 Microarray 실험을 수행

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

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19.4K 조회수.  University of Minnesota. 사용자 정의 microRNA의 microarray 실험을 수행 간단한 절차 설명합니다. 단계, RNA, RNA 라벨링 및 참조 DNA를 분리 microarrays에 샘플을 hybridizing, microarrays를 검색, quantifying 및 하이브리드화 신호를 분석 등이 있습니다.

비디오: Performing Custom MicroRNA Microarray Experiments

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Microarray 실험에 대한 Gibberella zeae Ascospore 생산 및 수집.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

연구

생물학

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

전기 생리학 실험을위한 인공 Bilayers의 준비

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

로블롤리 파인 PtGen2 cDNA Microarray 처리

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Zebrafish Oligonucleotide Microarray의 플랫폼을 사용하여 글로벌 유전자 발현 분석

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray 분석을위한 Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

CompoZr 맞춤 아연 손가락 Nucleases와 게놈 편집 (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

마이크로 RNA<em&gt; 현장에서</em&gt; 포르말린 고정 신장 조직에 대한 하이브리드

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

Vivo에서 심장 관련 예측에 관한-스폰지의 Nanovector 납품

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

마이크로RNA 클러스터 네트워크 분석을 위한 CRISPR 유전자 편집 도구

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

Performing Muscle Cryoinjury on a Zebrafish

For performing the myomere cryoinjury on zebrafish, use a custom manufactured stainless steel cryoprobe. To avoid frostbite while handling the probe during the procedure, insert the probe handle into a pipette tip and secure it by wrapping it with tape. Before beginning with the cryoinjury procedure, prepare a beaker for the anesthesia working solution, a spoon for handling the fish, a moist sponge, and a tank with system water for post-procedure recovery of the fish.Prepare the anesthesia stock solution following the steps shown on the screen. Then add four milliliters of the prepared stock solution to 100 milliliters of system water in a beaker to prepare the tricaine working solution. To begin the cryoinjury procedure, immerse the probe in liquid nitrogen for a minimum of three minutes.Wet the sponge in system water and place it on a flat surface. Then one to two minutes after transferring a single adult zebrafish into the tricaine working solution, confirm its unresponsiveness by touching the fish gently with the spoon. Place the anesthetized fish onto the wet sponge.Locate the caudal peduncle posterior to the anal fin and anterior to the caudal fin. At this stage, remove the cryoprobe from the liquid nitrogen. Shake the probe gently to ensure no residual liquid nitrogen remains on the tip.Place the edge of the spatula perpendicular to the body on the caudal peduncle. Keep the probe in this position for six seconds without applying pressure, before releasing the cryoprobe from the tissue. Then transfer the fish into the system water in the recovery tank.Monitor the fish while it resumes breathing and swimming after waking up from the anesthesia. If opercular movements do not occur after 30 seconds, stimulate the fish by pipetting system water into the gills until the animal initiates respiration by itself. Video recording of control and cryoinjured fish at one day post-injury or DPCI revealed that although the cryoinjured fish swam less actively, they did not display any abnormal movements, such as swirling, convolution, or reduced equilibrium.In the husbandry system, their position in the tank and food intake was similar to those of the uninjured fish. As observed at 5 DPCI, normal behavior was still persistent indicating the cryoinjury procedure did not severely affect the wellbeing of the animals.