Name: non-radioactive in situ hybridization protocol applicable for norway spruce and a range of plant species
Uploaded: 2009-04-17T00:00:00
Duration: 11 min 56 s
Description: Watch this Scientific Journal Video about Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species at JoVE.com

我们非常感谢志愿扬声器的声音安德斯Bovin提供我们的电影音乐和迈克尔埃利奥特股市。提供了支持卡尔Trygger基金会战略研究计划农业功能基因组学在瑞典农业科学院，瑞典研究理事会（VR）的大学（AgriFunGen），瑞典研究理事会和环境，农业科学，空间规划（FORMAS ）。

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+in+plants.">In situ hybridization in plants.</a> Molecular Plant Patology: a practical approach. , (1991).</li><li>Leitch, A. R., Schwarzacher, T., Jackson, D., Leitch, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization: a practical guide. , (1994).</li><li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+flower+development+in+Arabidopsis.">Early flower development in Arabidopsis.</a> Plant Cell. 2 (8), 755-767 (1990).</li><li>Azevedo, H., Lino-Neto, T., Tavares, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+high-quality+RNA+isolation+from+needles+of+adult+maritime+pine+trees.">An improved method for high-quality RNA isolation from needles of adult maritime pine trees.</a> Plant Molecular Biology Reporter. , 333-338 (2003).</li><li>Sundstrom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+active+in+developing+pollen+cones+of+Norway+spruce+(Picea+abies)+are+homologous+to+the+B-class+floral+homeotic+genes+in+angiosperms.">MADS-box genes active in developing pollen cones of Norway spruce (Picea abies) are homologous to the B-class floral homeotic genes in angiosperms.</a> Dev Genet. 25 (3), 253-266 (1999).</li><li>Tandre, K., Svenson, M., Svensson, M. E., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+gene+structure+and+activity+in+the+regulation+of+reproductive+organ+development+of+conifers+and+angiosperms.">Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms.</a> Plant J. 15 (5), 615-623 (1998).</li><li>Winter, K. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+reveal+that+gnetophytes+are+more+closely+related+to+conifers+than+to+flowering+plants.">MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants.</a> Proc Natl Acad Sci U S A. 96 (13), 7342-7347 (1999).</li><li>Savard, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+and+nuclear+gene+sequences+indicate+late+Pennsylvanian+time+for+the+last+common+ancestor+of+extant+seed+plants.">Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.</a> Proc Natl Acad Sci U S A. 91 (11), 5163-5167 (1994).</li><li>Jack, T., Brockman, L. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeotic+gene+APETALA3+of+Arabidopsis+thaliana+encodes+a+MADS+box+and+is+expressed+in+petals+and+stamens.">The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens.</a> Cell. 68 (4), 683-697 (1992).</li><li>Bowman, J. L., Smyth, D. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+directing+flower+development+in+Arabidopsis.">Genes directing flower development in Arabidopsis.</a> Plant Cell. 1 (1), 37-52 (1989).</li><li>Kramer, E. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+genetic+mechanisms+controlling+petal+development.">Evolution of genetic mechanisms controlling petal development.</a> Nature. 399 (6732), 144-148 (1999).</li><li>Sundstrom, J., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conifer+reproductive+development+involves+B-type+MADS-box+genes+with+distinct+and+different+activities+in+male+organ+primordia.">Conifer reproductive development involves B-type MADS-box genes with distinct and different activities in male organ primordia.</a> Plant J. 31 (2), 161-169 (2002).</li><li>Carlsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+altered+expression+of+a+large+number+of+nuclear+genes+in+developing+cytoplasmic+male+sterile+Brassica+napus+flowers.">Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers.</a> Plant J. 49 (3), 452-462 (2007).</li><li>Teixeira, R. T., Farbos, I., Glimelius, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+levels+of+meristem+identity+and+homeotic+genes+are+modified+by+nuclear-mitochondrial+interactions+in+alloplasmic+male-sterile+lines+of+Brassica+napus.">Expression levels of meristem identity and homeotic genes are modified by nuclear-mitochondrial interactions in alloplasmic male-sterile lines of Brassica napus.</a> Plant J. 42 (5), 731-742 (2005).</li><li>Ying, S., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+of+in+situ+hybridization.">The technique of in situ hybridization.</a> Methods in Molecular Medicine. 56, 263-283 (2001).</li></ol>

两方面的P.冷杉协议进行了优化比较的A.拟南芥/ B油菜协议，治疗核糖核酸酶和蛋白酶K的浓度。的RNase去除背景信号，从而增加了信号的特异性17。蛋白酶K的治疗是需要通透的组织，使探针可以进入和杂交利益的RNA池。 DAL13反义探针了一个更高的信号无RNase的治疗（图3C，E，G）相比，用RNase处理的部分为30分钟（图2D，F和H） 。由于核糖核酸治疗没有增强信号，这是从协议中删除。蛋白酶K的治疗进行了测试三种不同的浓度; 1，3和5微克/毫升。在P 的情况下冷杉一个低或无信号，观察使用1μg/ ml的蛋白酶K（图3C - D）。一个强烈的信号，两个3微克/毫升（图3E - F）和5微克/毫升（图3G - H）的蛋白酶K由于没有明显的差异，信号强度时发现使用5微克/毫升的蛋白酶K 3微克/毫升相比，我们选择使用我们的协议在3微克/毫升。很可能需要较高的蛋白酶K含量的体育冷杉相比，雄球花的A.拟南芥和 B。 油菜花芽是由于更紧凑的组织水平较高的酚类物质和多糖。 在固定和嵌入的A.之间的一些分歧拟南芥/ B甘蓝型油菜的协议和在体育冷杉协议是显而易见的。是固定的利益的组织提供RNA的保留，这是关键的一步，因为不好固定材料可能会低或检测不到信号，即使mRNA的是非常丰富的。组织脱水，清除和嵌入式蜡在逐渐变化，以避免组织损伤，并在一些协议0.85％的NaCl添加乙醇脱水，以进一步避免收缩和肿胀的细胞 3 。嵌入过程中的脱水步骤，可在冰上进行，保持在最低RNase活性。在体育冷杉协议的4％多聚甲醛修复方案包含0.25％的戊二醛，这是应该给予更好的 RNA保留17尽管有些人认为它可能没有什么区别3。后切片材料固定到幻灯片和预处理（即脱蜡，水化，透，处理，以减少非特异性结合的探针，最后脱水）杂交前。 信号的发展，整个检测过程在这里提出的协议可在普通实验室进行，通常在1-3天，超过背景信号之间的比例可以不断地进行监测。辛标记的探针通常会更鲜明的信号相比，放射性标记的探针，更稳定，可在连续的实验重用。另一方面，灵敏度降低相比，17个放射性探针原位杂交检测表达一定的mRNA的具体使用探针。射频标签是一个强大的和敏感的标记方法，但它需要处理的放射性物质，它需要几个星期前可检测的结果。另一方面抗原标记的探针，更稳定，危险性较小，需要接触到一个只有17几天的检测介质。因此，抗原标记方法是目前的主导。最关键的一步，不管协议是探头的设计。应谨慎，以确保探针具有高特异性和，是与高品质UTPs标记的。有时，杂交温度和/或反应的长度必须为特定的探头调整。 修改后的协议，基于高辛标记探针将促进mRNA的表达本地化， 同时在从A不等的物种拟南芥到 P 冷杉 ，应稍作调整在其他植物中使用。该协议，雄球花旁，也进行了测试在不同阶段的营养芽和合子和体细胞胚胎从P.冷杉与良好的结果（未公布结果;高本汉等 ）。

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Acetic anhydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6404-200ml</td>
<td>minimum 98%</td>
</tr>
<tr>
<td>Acrylamide, linear</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>9520</td>
<td>&nbsp;</td>
<tr>
<td>Anti-DIG-antibodies</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Blocking reagent</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11 175 041 910 or 11 096 176 001</td>
<td>&nbsp;</td>
<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A7030-50g</td>
<td>minimum 98%</td>
</tr>
<tr>
<td>Deionized formamide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Denhardts solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2532-5ml</td>
<td>&nbsp;</td>
<tr>
<td>DEPC, diethylpyrocarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dextran sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DIG RNA Labeling Kit (SP6/T7)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11175025910</td>
<td>&nbsp;</td>
<tr>
<td>Glutaraldehyde (25%)</td>
<td>&nbsp;</td>
<td>Histolab products AB</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>9510G</td>
<td>&nbsp;</td>
<tr>
<td>Histoclear II</td>
<td>&nbsp;</td>
<td>Histolab products AB</td>
<td>&nbsp;</td>
<td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td>
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<tr>
<td>Histowax</td>
<td>&nbsp;</td>
<td>Histolab products AB</td>
<td>&nbsp;</td>
<td>Histowax or Paraplast can be used for embedding.</td>
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<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6148-500g</td>
<td>&nbsp;</td>
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<td>Paraplast plus</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P3683-1kg</td>
<td>Histowax or Paraplast can be used for embedding.</td>
</tr>
<tr>
<td>Phusion&#x2122; High-Fidility DNA Polymerase</td>
<td>&nbsp;</td>
<td>Finnzymes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Probe-on Plus slides</td>
<td>&nbsp;</td>
<td>Fischer Scientific</td>
<td>22-230-900</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2308-5mg</td>
<td>&nbsp;</td>
<tr>
<td>RNase A</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R5503-100mg</td>
<td>&nbsp;</td>
<tr>
<td>Tissue-clear</td>
<td>&nbsp;</td>
<td>Sakura Finetek Europ&#233; BV</td>
<td>&nbsp;</td>
<td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td>
</tr>
<tr>
<td>Triethanolamine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1377-100ml</td>
<td>minimum 98%</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>use your standard product in the lab</td>
<td>&nbsp;</td>
<td>Use one or both of Triton X-100 and Tween-20.</td>
</tr>
<tr>
<td>tRNA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>83853-100mg</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>use your standard product in the lab</td>
<td>&nbsp;</td>
<td>Use one or both of Triton X-100 and Tween-20.</td>
</tr>
<tr>
<td>Western Blue</td>
<td>&nbsp;</td>
<td>Promega Corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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non-radioactive in situ hybridization protocol applicable for norway spruce and a range of plant species

高通量表达分析技术提供给科学家溢出的表达谱，但他们在组织特异性表达的决议，是因为在剖析个别组织的问题有限。表达数据需要进一步证实，表达用原位杂交，采用的技术，例如本地化细胞特异性mRNA的表达模式与补充。 原位杂交方法是费力，费时，往往需要广泛的优化取决于物种和组织。 原位实验木本物种，如针叶树挪威云杉（云杉冷杉）执行比较困难。在这里， 我们目前在修改后的辛原位杂交协议，这是快速，适用的植物种类广泛 ，包括体育冷杉 。短短几年的调整，包括改变的核糖核酸酶治疗和蛋白酶K的浓度，我们可以使用该协议的研究组织特异性表达的同源基因在男性生殖器官之一裸子植物和两个被子植物 ; P。 冷杉属，拟南芥和甘蓝型油菜 。该协议工作同样适用物种和基因研究。AtAP3和BnAP3螺纹A 中的第二个和第三个花器官观察拟南芥和 B。 油菜和DAL13 P.雄球花microsporophylls 冷杉 。 为 P 冷杉蛋白酶K的浓度，用于permeablize组织，必须增加至3克/毫升，而不是1克/毫升，可能是由于更紧凑的组织和更高水平的酚类物质和多糖。所有种类的核糖核酸酶治疗被删除，由于没有相应增加特异性信号强度降低。通过比较同源基因的组织特异性表达模式，从开花植物和针叶树，我们证明了DIG原位协议提出，只有分钟的调整，可应用于广泛的植物物种。因此，这个协议避免了双方广泛的物种特定的优化和赞成DIG标记的探针的放射性标记的探针费力的使用。我们已经选择了，说明在我们的电影协议的技术要求高的步骤。 高本汉安娜和珍妮卡尔松同等贡献这项研究。 通讯作者：安娜Anna.Karlgren @ ebc.uu.se和Jens楼松德斯特伦Jens.Sundstrom @ vbsg.slu.se高本汉

Watch this Scientific Journal Video about Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species at JoVE.com

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

我们描述了一个修改的辛<em&gt;原位</em&gt;杂交协议，它是快速和适用的植物种类广泛，包括挪威云杉。短短几年的调整，包括改变的核糖核酸酶治疗和蛋白酶K的浓度，该协议可用于在不同的组织和物种的研究。

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

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Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

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

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

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</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 ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

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

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

快速荧光<em&gt;原位</em&gt;对于Xist的RNA杂交组合议定书组蛋白修饰免疫荧光X染色体失活

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...