저자는 수년 동안 프로토콜을 향상 모든 자비스 실험실 구성원을 감사드립니다.

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우리는 공개 할게 없다.

mRNA의 표현의 원위치 하이브리드화의 방사성 널리 지방 조직 조직, 세포 유형과 뇌의 기능적 활동 2-5,10,12-14 공부를 포함하여 여러 용도에 사용됩니다. 나중에 사용할 누구의 mRNA 표현 뇌의 신경 활동이 증가, 종종 불리는 활동 종속적인 유전자 또는 즉각 조기 유전자에 따라 좌우 유전자에 있습니다. hatchlings / neonates, 청소년을 포함한 여러 연령대, 여러 조직에서, 뇌, 피부 및 근육을 포함하여, 이러한 용도로, 우리의 방법은 포유류 (예 : 인간), 생선 및 양서류, 조류를 포함한 여러 종류에 걸쳐 적용되었습니다 , 성인, 그리고 이곳 전체 배아 부분에 2,3,5,15-17. 우리의 프로토콜의 특징은 다음과 같습니다 : (1) 그것은 해부 학적 특이성과 양적 특이성 사이의 균형을 생산하고 있습니다. x-선 필름에서 유전자 발현을 계량하기 위해, 우리는 이미지의 디지털 사진 (. 예 : 그림 1A 및 1B)를 받아, 검사하고 사진에게 사용관심 지역에서 픽셀 밀도를 측정하고 조직 외부 필름에 배경 수준을 빼야하지만 여전히 유리 슬라이드 2,4에서하는 toshop (어도비) 히스토그램 기능. 세포 수준에서 표현을 계량하기 위해, 우리는 높은 배율 (; 그림 4 40-100X)에서 세포를 통해 실버 곡물의 이미지 가져가라. 우리는 다음 유리 슬라이드에 세포가없이 유사한 영역에서 배경 카운트를 뺍니다, 이미지에 은색 알갱이의 수를 계산하기 위해 NIH에서 웨인 Rasband하여 이미지 J의 문턱 및 측정 기능을 이용, 세포의 숫자로 나누면 셀 당 표현. 4,18의 값 (2) 그것은 동시에 100-200 슬라이드 처리를 허용, 미네랄 오일 목욕 만든 긴밀한 coverslip 인감으로 인해 상대적으로 높은 처리량이 될 수 있습니다. 구하는 표준의 원위치 하이브리드화 방법 parafilm, 매니큐어, 그리고 슬라이드는 공간을 많이 차지 다른 방법과 슬라이드를 봉인하는 데 오래 걸립니다 (3) 그것은 아주이다Dextran 황산과 하이브리드화 버퍼 2,13의 Denhardt의 솔루션으로 인해 낮은 풍요의 성적에 대한 민감한 (4) darkfield의 이미징 접근법은 4,5 해부 현미경에 darkfield 조명 아래에서 사진을 찍고으로 인해 높은 명암 이미지를 얻을. 또한,이 같은 활동에 의존 유전자 발현에서와 같이 유전자 발현의 작은 변화의 민감한 검출 19 특정 행동의 인식 및 생산 기간 동안 활성화된 특정 뇌 영역을 식별할 수 있습니다. 비 방사성 프로토콜에 상대적으로 제한 후자는 세포 해상도와 세포 내에서 mRNA의 위치에 명확하다고하며, 유제와 협력하는 것은 조작과 빛을 매우 민감합니다. 이 같은 조직 10,17,20에서 하나 이상의 유전자의 mRNA 발현에 레이블을 위해 현장 하이브리드화 이러한 비 방사성 등 다른 방식으로 우리의 방법을 결합하는 것이 가능합니다. 그것은 immunocytochemistry와 조합하여 사용할 수 있습니다위해서는 동일한 시료에 RNA와 단백질 표현 모두에 레이블을 특정 세포 유형 10와 mRNA가 공동 집중하다. 이러한 이중 라벨 실험에 필요한 프로토콜 수정은 인용된 참고 문헌에 설명되어 있습니다. 요약하면, 우리의 접근 방법은 지역 조직, 조직 기능적 활동 및 유전자 기능을 이해하기 위해 타이밍과 유전자 발현의 세포 위치의 이해를 용이하게합니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 아세트산 무수물 </td><td> VWR </td><td> MK242002 </td><td></td></tr><tr><td> 클로로포름 </td><td> VWR </td><td> BDH1109 </td><td></td></tr><tr><td> Cresyl 보라색 아세트산 </td><td> 시그마 </td><td> C5042 </td><td></td></tr><tr><td> 그라 이오 스탯 </td><td> 써모 과학 </td><td> Microm HM550 </td><td></td></tr><tr><td> Deionized 포름 아미드 </td><td> 시그마 </td><td> F9037 </td><td></td></tr><tr><td> DTT </td><td> Promega </td><td> P1171 </td><td> 100mM </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 시그마 </td><td> ED </td><td></td></tr><tr><td> 금형을 포함 </td><td> VWR </td><td> 15160-215 </td><td></td></tr><tr><td> 피셔 브랜드 Superfrost 플러스 슬라이드 </td><td> 피셔 과학 </td><td> 22-034-979 </TD&gt; <td></td></tr><tr><td> 포름 알데히드 </td><td> VWR </td><td> BDH0506-4LP </td><td></td></tr><tr><td> 포름 아미드 </td><td> 시그마 </td><td> F7508 </td><td></td></tr><tr><td> GENECLEAN 키트 </td><td> Q-바이오 유전자 </td><td> 1001-200 </td><td></td></tr><tr><td> 코닥 BioMax MR 필름 </td><td> 시그마 </td><td> Z350370 </td><td></td></tr><tr><td> 코닥 NTB 에멀젼 </td><td> Carestream 건강 </td><td> 8,895,666 </td><td></td></tr><tr><td> 코닥 프로 페셔널 개발자 D19 </td><td> 코닥 </td><td> 1,462,593 </td><td></td></tr><tr><td> 코닥 전문 해결사 </td><td> 코닥 </td><td> 1,971,746 </td><td></td></tr><tr><td> β-메르 캅 토 에탄올 </td><td> Calbiochem </td><td> 444,203 </td><td></td></tr><tr><td> 미네랄 오일 </td><td> VWR </td><td> IC15169491 </td><td></td></tr><tr><td> NaOH </td><td> VWR </td><td> SX0600-1 </td><td></td></tr><tr><td> Paraformaldehyde </td><td> 시그마 </td><td> 76,240 </td><td></td></tr><tr><td> 폴리 </td><td> Invitrogen </td><td> POLYA.GF </td><td></td></tr><tr><td> rATP </td><td> Promega </td><td> P1132 </td><td> 10mM </td></tr><tr><td> rCTP </td><td> Promega </td><td> P1142 </td><td> 10mM </td></tr><tr><td> rGTP </td><td> Promega </td><td> P1152 </td><td> 10mM </td></tr><tr><td> RNasin </td><td> Promega </td><td> N2111 </td><td> 40Units/μl </td></tr><tr><td> S 35 UTP </td><td> PerkinElmer </td><td> NEG039C001MC </td><td></td></tr><tr><td> 안전 해결 솔루션 </td><td> 안전 연구 제품 국제 해결 </td><td> 111,177 </td><td></td></tr><tr><td> 아세트산 나트륨 </td><td> 시그마 </td><td> S7899 </td><td> 3M </td></tr><tr><td> 나트륨 인산염 이염 </td><td> 시그마 </td><td> S3264 </td><td></td></tr><tr><td> 나트륨 인산염 일염기의 </td><td> 시그마 </td><td> S3139 </td><td></td></tr><tr><td> SP6RNA 중합 효소의 </td><td> Promega </td><td> P1085 </td><td></td></tr><tr><td> 금속 선반을 염색법 </td><td> 전자 현미경 과학 </td><td> 70312-54 </td><td></td></tr><tr><td> T7 RNA 중합 효소의 </td><td> Promega </td><td> P2075 </td><td></td></tr><tr><td> 조직 - 테크 10월 </td><td> 사쿠라 </td><td> 4583 </td><td></td></tr><tr><td> 5 배 전사 버퍼 </td><td> Promega </td><td> P1181 </td><td></td></tr><tr><td> Triethanolamine </td><td> VWR </td><td> IC15216391 </td><td></td></tr><tr><td> 트리스-HCL (1 M, 산도 8.0) </td><td> VWR </td><td> 101449-446 </td><td></td></tr><tr><td> tRNA </td><td> 로슈 </td><td> 10109509001 </td><td></td></tr></tbody></table> 솔루션 : <ol><li> riboprobes로 만들기위한 시약 : 1.5 μl의 DNA 템플릿 (0.2-0.3 μg / μl), 5 배 최적화된 전송 버퍼의 2 μl, 100mM DTT 1 μl (Promega에서 중합 효소와 함께 제공, 믹스잘 상온에서), RNasin의 0.3 μl (40 대 / μl), AGC ribonucleotide 믹스 솔루션, S 35 UTP 3.5 μl, 1 μl RNA 중합 효소의 1.5 μl. nuclease없는 물을 갖춘 10 μl까지 와요. </li><li> AGC의 ribonucleotide 믹스 솔루션 : 10mM ATP, GTP와 함께 CTP의 동일한 금액을 섞는다. </li><li> 40 μl RNase와 DNase 무료 물, 3M의 아세트산 나트륨 및 100 % EtOH 125 μl의 5 μl : 무료 S 35 UTP를 제거하는 EtOH 침전에 대한 아세트산 나트륨 용액. </li><li> 하이브리드화 버퍼 (10ml 주식) : 100 % deionized 포름 아미드, 5M NaCl 600 μl, 1M 트리스-HCL (산도가 = 8.0)의 5 ML, 0.5M EDTA (에틸렌 다이아 민 테트라 초산) 240 μl (산도 = 8.0), 100x Denhart의 솔루션 100 μl, 1M DTT, 20mg/ml tRNA, 20 밀리그램 / ML 폴리 125 μl, 그리고 나트륨 Dextran 황산의 1g 250 μl의 100 μl. 10 ML에 대한 전체 볼륨을 가지고 DEPC-처리된 물을 추가합니다. 모든 나트륨 dextran 황산이 녹아 때까지 55 ° C에서 부화 후 적극적으로 흔들. 에 하이브리드화 버퍼를 저장~ 6 개월 좋다 -20 ° C,. </li><li> 4 % paraformaldehyde 용액을 버퍼 : paraformaldehyde를 위해 지정, 50 ~ 열이 ° C 따뜻한 접시에 감동하면서 플라스크에 760 ML 증류수에 paraformaldehyde 40 G를 추가합니다. paraformaldehyde를 해산하기 위해 10N NaOH 320 μl를 추가합니다. (~ 10 분) 용해 후, 10X PBS 100 ML을 추가하고 1리터 때까지 증류수로 볼륨을 제기하셨습니다. 저어과 paraformaldehyde가 해소되기 전까지 솔루션을 가열. 산도는 7.4이어야합니다. </li><li> 아세틸화 버퍼 : triethanolamine의 13.6 ML 플러스 증류수 1 리터에 아세트산 무수물의 2.52 ML. </li><li> 20x SSPE 솔루션 : 3M NaCl, 200 MM 아뇨 2 PO 4-H 2 O 및 증류수 200 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산). 10N NaOH로 pH를 7.4에 대한 해결 방법을 조정합니다. </li><li> 10X PBS 버퍼 : NaCl의 80g, KCl의 2.0g, 나 2 HPO 4 14.4g과 KH 2 2.4g 증류수에 PO 4. 산도 7.0에 대한 솔루션을 조정하고 1 라이트로 전체 볼륨을 가지고R. </li><li> 두번째 세차 솔루션 : 0.1 % β-메르 캅 토 에탄올과 2X SSPE에서 50 %의 포름 아미드 </li><li> Cresyl 바이올렛 아세트산 용액 : 수돗물의 3% cresyl 보라색 아세트산 (증류수 좋은 염색법을 방지), 실온에서 플라스크에 감동과 함께 하룻밤 해산. 1mm 워트 먼지 필터 종이와 Buchner 퍼널을 통해 진공 흡입으로 필터링합니다. </li></ol>

radioactive in situ hybridization for detecting diverse gene expression patterns in tissue

유전자가 표현되는 타이밍, 레벨, 세포 현지화 및 세포 유형을 안다는 건 유전자의 기능에 대한 우리의 이해에 기여한다. 이러한 기능을 각각의 세포 내에서 mRNAs에 원위치 하이브리드화에서 함께 수행할 수 있습니다. 여기 클레이튼 외. (1988) 1, 특히 성인 척추 두뇌 2-5 위해, 여러 해 동안 우리가 실험실에서 성공적으로 작동되었습니다에서 수정된 원위치 하이브리드화 방법에 방사성을 제시. 대상 시퀀스로 긴 보완 RNA (cRNA) 프로브는 낮은 풍요의 성적 6,7의 검출을 허용합니다. cRNA 프로브로 방사성 세포핵의 설립이 낮은 풍부한 성적 증명서 및 정량 분석​​에 대한 검출 감도를 허용 역시 가벼운 민감한 X 선 필름이나 조직을 통해 코팅 에멀젼에 의해. 이러한 검색 방법은 타겟 유전자 표현의 장기 기록을 제공합니다. 비 방사능 탐사 필로폰과 비교이러한 쫄지마-라벨 등 ODS, 방사성 프로브 하이브 리다이 제이션 방식은 낮은 풍요로움의 성적 증명서를 감지할 수 HRP-항체 및 / 또는 TSA 키트를 사용하여 여러 증폭 단계를 필요로하지 않습니다. 따라서이 방법은 정량 분석​​을위한 신호 강도와 타겟 mRNA 금액 사이의 선형 관계를 제공합니다. 그것은 동시에 100-200 슬라이드를 처리하실 수 있습니다. 그것은 배아의 여러 발달 단계를 위해 잘 작동합니다. 배아 세포가 어려운 조직 섹션에있는 세포 집단 사이의 경계를 확인하고, 세포 사이에 덜 결합으로, 어른 조직보다 더 깨지기 때문에 유전자 발현의 대부분은 발달 연구 부분에 전체 배아 및 비 - 방사성 방법 8,9를 사용합니다. 반대로 감성의 더 큰 범위로 인해 우리의 방사성 접근은 쉽게 인구 사이의 경계를 확인하고, 조직 지역 간의 유전자 발현의 해상도에서 높은 콘트라스트를 얻을 수 있습니다. 이 방법을 사용하여 연구자가 속한새로 확인된 유전자의 가능한 의미, 그리고 더 관심이 유전자의 기능을 예측했다.

Watch this Scientific Journal Video about Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue at JoVE.com

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

이 프로토콜이 성공적으로 양적 척추 종의 여러 조직 유형의 수준과 mRNA의 표현의 공간적 패턴을 감지하는 데 사용됩니다. 방법은 낮은 풍요로움의 성적 증명서를 감지하고 동시에 슬라이드 수백 처리 가능합니다. 우리는 예제와 같은 조류 배아 뇌의 형성의 표현 프로파일을 사용하여이 프로토콜을 제시한다.

방사성 현장에서</em조직에서 다양한 유전자 발현 패턴을 검출> 하이브리드화

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

radioactive-situ-hybridization-for-detecting-diverse-gene-expression

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Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

15.5K 조회수.  Duke University. 이 프로토콜이 성공적으로 양적 척추 종의 여러 조직 유형의 수준과 mRNA의 표현의 공간적 패턴을 감지하는 데 사용됩니다. 방법은 낮은 풍요로움의 성적 증명서를 감지하고 동시에 슬라이드 수백 처리 가능합니다. 우리는 예제와 같은 조류 배아 뇌의 형성의 표현 프로파일을 사용하여이 프로토콜을 제시한다.

비디오: Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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생물학

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

비 - 방사성 현장에서 하이브 리다이 제이션 노르웨이 스프루스에 대한 해당 프로토콜과 식물 종의 범위

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

높은 처리량 현장에서</em태아 마우스 로우어 Urogenital 트랙트의 mRNA의 표현 패턴을 특성화> 하이브리드화 방법

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA 형광하여 단일 세포 유전자 전사의 분석<em&gt; 현장에서</em&gt; 하이브리드 (물고기)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

높은 해상도 전체 마운트<em&gt; 제자리에서</em유전자 발현 및 기능을 연구하는 제브라 피쉬 배아에서&gt; 하이브리드

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

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 ...

방사성 현장에서 하이브리드화