单细胞基因转录的RNA荧光分析<em在原位</em>杂交（FISH）
1Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, Faculty of Health Sciences,University of Copenhagen, 2Department of Infectious Diseases,Copenhagen University Hospital (Rigshospitalet), 3Institute of Infection and Immunology Research, School of Biology,University of Edinburgh
Summary
荧<em在原位</em>杂交（FISH），以确定在单个细胞中的mRNA转录如同时转录中的一个以上的成员的允许多基因活性分析<em> VAR</em>多基因家族<em>恶性疟原虫</em>受感染的红细胞<sup> 1</sup>。该技术的适应性和可用于不同类型的基因，细胞和有机体。
Abstract
恶性疟原虫感染的红细胞（IE）在疟疾感染的人脐静脉内皮受体介导的黏附表达的PfEMP1 由 var基因编码的蛋白变异。
单倍体P.恶性疟原虫基因组中藏着约60个不同的变种基因，其中只有一个被认为是转录每一次细胞在血液的感染阶段。如何实现这种相互排斥的变种基因的转录调控还不清楚，因为是个人var基因的鉴定或小组var基因与不同的受体和后果的差分结合的临床结果P.恶性疟原虫感染。近日，被称为相互排斥的转录模式提出了质疑，根据个人P.转录分析恶性疟原虫转录鉴定单INF的ected红细胞细胞利用RNA 荧光原位杂交（FISH）分析VAR的寄生虫P.单个核基因转录恶性疟原虫即：1。
在这里，我们提出了一个详细的协议进行分析变种基因的转录的RNA-FISH方法在单核的P.恶性疟原虫感染人类的红细胞。该方法是基于对使用地高辛和生物素标记的反义RNA探针使用的TSA加荧光调色板系统2（Perkin Elmer公司制），微观分析和新鲜选择P.恶性 IE浏览器。 原位杂交法可以用来监测转录的各种基因表达和调节过程中的不同阶段的P.恶性疟原虫生命周期和适应其他疟疾寄生虫物种和其他生物体和细胞类型。
Protocol
1。新选择受感染的红细胞的生成对于这个测定法，获得最好的结果是当使用新鲜选择培养表面表达的PfEMP1蛋白。在这个特殊的实验3D7 P.恶性疟原虫谱系选择使用特定的抗体，如前面描述的1。 第1天嘉实200μl的包装血细胞从P.恶性疟原虫培养含有2-5％的IE后期通过离心分离，在800×g离心8分钟，在室温下。 血液沉淀重新悬浮…
Representative Results
图1示出的主要步骤的流程图，和持续时间的RNA FISH方法。 一系列有代表性的图像，保存差染mRNA的FISH实验采用单P.疟原虫 IE浏览器在图2中所示。细胞内的原虫有一个小的（1-1.5微米直径）的原子核，用DAPI染成蓝色。二十四小时后，红细胞的入侵P.恶性疟原虫裂殖过程中， 即细胞核分裂，就开始了。优化FISH检测的变种基因转…
Discussion
RNA FISH分析，如Northern印迹和RT-PCR的方法，允许特定的mRNA转录在单细胞水平上的歧视。这使得有可能区分转录活性和非活动单元，在这个例子中，P.恶性疟原虫在人体红血细胞内寄生原虫。这种全细胞观察往往是必要的，并可能解开重要的和新颖的转录模式1。
虽然其他RNA FISH的方法已被描述4-10，出现了一个需要发展的一个新的和精致的协议，用于研究?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者想感谢迈克尔·Alifrangis和乌拉Abildtrup，基因分型的寄生虫和克里斯蒂娜·霍尔姆优秀的技术援助。这项工作是由霍华德休斯医学研究所（授予55005511），，Lundbeck公司基金（批准R9-A840）和尼尔斯·玻尔基金会。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Acetic Acid | Sigma/Aldrich | 338826-100ml | |
| Albumax media: RPMI 1640 Glutamine solution | Lonza | BE12-115F | 500 ml RPMI 1640
5 ml glutamine solution |
| Albumax media: Gentamycin sulphate Albumax-solution | Lonza | BE02-012E | 2.5 ml gentamycin sulphate
50 ml Albumax-solution |
| Albumax-solution: Hypoxanthine | Sigma-Aldrich | H9377 | 0.8 g Hypoxanthine |
| Albumax-solution: AlbuMAX II | Invitrogen | 11021-037 | 200 g Albumax |
| Albumax-solution: RPMI 1640 | Lonza | BE12-115F | 4 liter RPMI 1640
Dissolve with magnet at max. 50 °C. Filter sterilize and store at -20 °C in aliquots. |
| Amberlite | Sigma | A5710-110G | Resin to deionize formamide. |
| Anti-biotin goat pAb Peroxidase Conjugate | Calbiochem | 203206 | |
| Anti-Dig antibody | Novus biologicals | NB100-41330 | |
| Anti-fade reagent with DAPI | Invitrogen | P36931 | The mounting media has to cure for 24 hr before sealing the slide completely. |
| Biotin RNA Labeling Mix | Roche | 11685597910 | |
| Camera digital | Nikon digital sight DC-F11 | ||
| Coverslips | Menzel-glaser | 631-1570 | |
| culture flask 25 cm2 Nunclon surface | Nunc | 156340 | |
| DEPC | Fluka/Sigma | 32490-100ml | 1 ml DEPC in 1000 ml deinonized water. Add a stir bar and stir for 12 hr. Autoclave for 30 min. |
| Digital camera | Nikon digital sight DC-F11 | ||
| Dynabeads Protein A | Invitrogen | 10002D | |
| DynaMaq-15 Magnet | Invitrogen | 123-01D | |
| Formamide bioultra 99 % | Fluka/Sigma | 47671-1l-F | Deionized formamide: 5 g of ion exchange resin per 100 ml of formamide. Stir 30 min. Filter through Whatman paper. |
| Gelatine 0.75% solution: Gelatine | Sigma-Aldrich | G2500 | 3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 °C to dissolve. Filter sterilize when 56 °C. Store at -20 °C in aliquots. |
| Gelatine 0.75% solution: RPMI 1640 | Lonza | BE12-115F | 3.75 g gelatine in 500 ml RPMI 1640. Heat to 56 °C to dissolve. Filter sterilize when 56 °C. Store at -20 °C in aliquots. |
| Glutamine solution: L-glutamin | Sigma-Aldrich | G3126 | 14.6 g L glutamine in 500 ml 0.9 % NaCl. Dissolve, filter sterilize and store at -20 °C in aliquots. |
| Glutamine solution: HCl | Sigma | H1758 | 14.6 g L glutamine in 500 ml 0.9 % NaCl. Dissolve, filter sterilize and store at -20 °C in aliquots. |
| Hybridization solution: Formamide Bio ultra 99% SSC 20x | Fluka/Sigma | 47671-1l-F | Total 20 ml, keep frozen at -20 °C in aliquots 10 ml deionized formamide |
| Hybridization solution: Denhardt’s 50x concentrate | Sigma | D2532 | 5 ml 20xSSC |
| Hybridization solution: Yeast tRNARoche blocking reagent | Sigma | R-6750 | 2 ml 50x Denhardt’s 250 μl 20 mg per ml yeast tRNA |
| Hybridization solution: Salmon sperm DNA | Fluka/Sigma | 31149-106GF | 0.4 g Roche blocking reagent 1 ml of 10 mg per ml salmon sperm DNA (Critical: denature salmon spermDNA at 96 °C for 5 min before adding to the hybridization solution) |
| Hybridizer ThermoStar 100 HC4 | Quantifoil Instruments GmbH | 1004-0011 | Can be replaced by hybridization oven and RNase free hybridization chambers padded with DEPC water. |
| Immersion oil UV transparent fluorescence free | Sigma | 10976-1EA | |
| Immunofluorescence or confocal microscope | Nikon D-Eclipse TE2000C | ||
| Nailpolish | Available in any drugstore | ||
| PBS 20x:NaCl
KCl Na2HPO4 x 2H2O KH2PO4 DEPC deionized H2O |
Ajust pH to 7.4
160 g 4 g 23 g 4 g 1 l |
||
| Paraformaldehyde 4 % | Fluka/chemika | 76240 | 4 g PFA in 80 ml PBS/DEPC. Heat to 65 °C until the PFA dissolves. Add 20 ml PBS, allow the solution to cool. Adjust the pH to 7.4. Filter. Store in aliquots at -20 °C. |
| Paraformaldehyde 4 %/ Acid acetic 5 % | 950 μl paraformaldhyde + 50 μl Acetic acid | ||
| Pepsin | Sigma/Aldrich | P7000 | |
| Peroxidase-conjugated anti-biotin | Calbiochem | 203206 | |
| RBC-wash media: RPMI 1640 Glutamine solution | Lonza | BE12-115F | 500 ml RPMI 1640 5 ml glutamine solution |
| RBC-wash media: Gentamycin sulfate | Lonza | BE02-012E | 2.5 ml gentamycin sulphate |
| RNase | Sigma/Aldrich | Make a 10 mg/ml stock solution. | |
| RNase free 1.5 ml tube | Ambion | AM12450 | |
| RNase Zap | Ambion | AM9780 | |
| Slides 4 wells 11 mm | Thermo Scientific | MENZXER306W | |
| SSC 20x: NaCl | Sigma/Aldrich | S9625 | 3 M NaCl (175 g/l) 0.3M Na3 citrate x H2O (88 g/l) Adjust to pH 7.0 with 1M HCl |
| SSC 20x: Sodium citrate dehydrate | Sigma/aldrich | W302600 | 3 M NaCl (175 g/l) 0.3M Na3 citrate x H2O (88 g/l) Adjust to pH 7.0 with 1M HCl |
| SSPE 20x: NaCl | Sigma/Aldrich | S9625 | 175.3 g NaCl |
| SSPE 20x: NaH2PO4 | Sigma/Aldrich | S0751 | 27.6 g NaH2PO4. |
| SSPE 20x:4 EDTA powder | 9.4 g EDTA powder Add DEPC water, adjust pH 7.4. Autoclave for 20 min |
||
| TNB buffer: TNT buffer | 10 ml TNT buffer | ||
| TNB buffer: Blocking reagent | 0.05 g Blocking reagent To dissolve the blocking reagent, heat the solution to 60 °C for one hour with stirring. Store at -20 °C. (Derived from the Perkin Elmer TSA-protocol.) |
||
| TNT buffer: Tris/HCl | Sigma | T1503 | 1M Tris/HCl, pH 8.0 |
| TNT buffer: NaCl | Sigma Aldrich | S9625 | 100 ml |
| TNT buffer: Tween20 | Sigma | 93773 | 5 M NaCl 30 ml 1 ml DEPC dH2O 869 Ml Adjust pH to 7.5 at room temperature. (Derived from the Perkin Elmer TSA-protocol.) |
| TSA plus Cyanine3/ Fluorescein system | Perkin Elmer | NEL753000IKT | Read the protocol from the TSA Plus Fluorescence Palette System carefully before starting the experiment. |
| Tubes 14 ml sterile | Almeco – CM LAB Aps | 91016 |
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Tags
Single-cell Gene TranscriptionRNA Fluorescent In Situ Hybridization (FISH)Plasmodium Falciparum Infected ErythrocytesPfEMP1 Protein VariantsVar GenesMutually Exclusive RegulationTranscription AssaysRNA-FISH MethodologyDigoxigenin-labeled Antisense RNA ProbesBiotin-labeled Antisense RNA ProbesTSA Plus Fluorescence Palette System
Citazione di questo articolo
Ronander, E., Bengtsson, D. C., Joergensen, L., Jensen, A. T. R., Arnot, D. E. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH). J. Vis. Exp. (68), e4073, doi:10.3791/4073 (2012).