Name: localization of odorant receptor genes in locust antennae by rna in situ hybridization
Uploaded: 2017-07-13T13:00:00
Description: Watch this Scientific Journal Video about Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization at JoVE.com

Эта работа поддерживается грантом Национального фонда естественных наук Китая (No.31472037). Упоминание торговых наименований или коммерческих продуктов в этой статье предназначено исключительно для целей предоставления конкретной информации и не подразумевает рекомендации.

ПРИМЕЧАНИЕ. Чтобы ограничить разложение РНК, подготовьте растворы, используя влажную автоклавированную дистиллированную воду (при 121 ° C в течение 60 минут), а также материалы с мокрым автоклавом. 1. Получение РНК ISH-антисмысловых и чувствительных зондов <ol><li> Усилени?...

<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=Molecular+basis+of+odor+coding+in+the+malaria+vector+mosquito+Anopheles+gambiae.">Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae.</a> Proc Natl Acad Sci U S A. 107 (9), 4418-4423 (2010).</li><li>Hassanali, A., Njagi, P. G., Bashir, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+ecology+of+locusts+and+related+acridids.">Chemical ecology of locusts and related acridids.</a> Annu Rev Entomol. 50, 223-245 (2005).</li><li>Schaeren-Wiemers, N., Gerfin-Moser, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+protocol+to+detect+transcripts+of+various+types+and+expression+levels+in+neural+tissue+and+cultured+cells:+in+situ+hybridization+using+digoxigenin-labeled+cRNA+probes.">A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labeled cRNA probes.</a> Histochemistry. 100 (6), 431-440 (1993).</li><li>Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+map+of+olfactory+receptor+expression+in+the+Drosophila+antenna.">A spatial map of olfactory receptor expression in the Drosophila antenna.</a> Cell. 96 (5), 725-736 (1999).</li><li>Yang, Y., Krieger, J., Zhang, L., Breer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+olfactory+co-receptor+Orco+from+the+migratory+locust+(Locusta+migratoria)+and+the+desert+locust+(Schistocerca+gregaria):+identification+and+expression+pattern.">The olfactory co-receptor Orco from the migratory locust (Locusta migratoria) and the desert locust (Schistocerca gregaria): identification and expression pattern.</a> Int J Biol Sci. 8 (2), 159-170 (2012).</li><li>Schultze, A., 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=The+co-expression+pattern+of+odorant+binding+proteins+and+olfactory+receptors+identify+distinct+trichoid+sensilla+on+the+antenna+of+the+malaria+mosquito+Anopheles+gambiae.">The co-expression pattern of odorant binding proteins and olfactory receptors identify distinct trichoid sensilla on the antenna of the malaria mosquito Anopheles gambiae.</a> PLoS One. 8 (7), e69412 (2013).</li><li>Xu, H., Guo, M., Yang, Y., You, Y., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+two+novel+odorant+receptors+in+the+locust+(Locusta+migratoria).">Differential expression of two novel odorant receptors in the locust (Locusta migratoria).</a> BMC Neurosci. 14, 50 (2013).</li><li>Saina, M., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+olfactory+receptor+expression+and+localization+in+Drosophila.">Visualizing olfactory receptor expression and localization in Drosophila.</a> Methods Mol Biol. 1003, 211-228 (2013).</li><li>Guo, M., Krieger, J., Gro&#223;e-Wilde, E., Mi&#223;bach, C., Zhang, L., Breer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+ionotropic+receptors+are+expressed+in+olfactory+sensory+neurons+of+coeloconic+sensilla+on+the+antenna+of+the+desert+locust+(Schistocerca+gregaria).">Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria).</a> Int J Biol Sci. 10 (1), 1-14 (2013).</li><li>You, Y., Smith, D. P., Lv, M., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broadly+tuned+odorant+receptor+in+neurons+of+trichoid+sensilla+in+locust,+Locusta+migratoria.">A broadly tuned odorant receptor in neurons of trichoid sensilla in locust, Locusta migratoria.</a> Insect Biochem Mol Biol. 79, 66-72 (2016).</li><li>Jiang, X., Pregitzer, P., Grosse-Wilde, E., Breer, H., Krieger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+two+"Sensory+Neuron+Membrane+Proteins"+(SNMPs)+of+the+desert+locust,+Schistocerca+gregaria+(Orthoptera:+Acrididae).">Identification and characterization of two "Sensory Neuron Membrane Proteins" (SNMPs) of the desert locust, Schistocerca gregaria (Orthoptera: Acrididae).</a> J Insect Sci. 16 (1), 33 (2016).</li><li>Angerer, L. M., Angerer, R. C., Wilkinson, D. G. <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+to+cellular+RNA+with+radiolabelled+RNA+probes.">In situ. hybridization to cellular RNA with radiolabelled RNA probes.</a> In situ hybridization. , 2 (1992).</li><li>Komminoth, P., Merk, F. B., Leav, I., Wolfe, H. J., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+35S-+and+digoxigenin-labeled+RNA+and+oligonucleotide+probes+for+in+situ+hybridization.+Expression+of+mRNA+of+the+seminal+vesicle+secretion+protein+II+and+androgen+receptor+genes+in+the+rat+prostate.">Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.</a> Histochemistry. 98 (4), 217-228 (1992).</li><li>Leary, J. J., Brigati, D. J., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+colorimetric+method+for+visualizing+biotin-labeled+DNA+probes+hybridized+to+DNA+or+RNA+immobilized+on+nitrocellulose:+Bio-blots.">Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.</a> Proc Natl Acad Sci U S A. 80 (13), 4045-4049 (1983).</li><li>Hallem, E. A., Ho, M. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+antenna.">The molecular basis of odor coding in the Drosophila antenna.</a> Cell. 117 (7), 965-979 (2004).</li><li>Jones, W. D., Nguyen, T. A., Kloss, B., Lee, K. J., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+conservation+of+an+insect+odorant+receptor+gene+across+250+million+years+of+evolution.">Functional conservation of an insect odorant receptor gene across 250 million years of evolution.</a> Curr Biol. 15 (4), R119-R121 (2005).</li></ol>

Трудно выполнить РНК ISH локализовать OR гены в антеннах насекомых, потому что уровни экспрессии генов OR, за исключением ORco , очень низки, и сохранение гистологических фрагментов антенн насекомых очень сложно. Кроме того, обнаружение TSA также очень сложно. Для решения этих проблем нео...

Насекомые-антенны, которые являются наиболее важными хемосенсорными органами, покрыты многими волоскоподобными структурами, называемыми сенсиллами, которые иннервируются обонятельными рецепторными нейронами (ОРН). На мембране ORN насекомых рецепторы одорантов (ORs), тип белка, содержащего семь трансмембранных доменов, экспрессируются с помощью корецептора (ORco) с образованием гетеромера, который функционирует как ионный канал 1 , 2 , 3 с одорантом. Различные ОР реагируют на различные комбинации химических соединений 4 , 5 , 6 . Саранча ( Locusta migratoria ) в основном полагается на обонятельные сигналы, чтобы вызвать важные формы поведения 7 . Осколки саранчи являются ключевыми факторами для понимания механизмов молекулярного обоняния. Локализация определенного гена ORG саранчи к нейронуМорфологически специфический тип сенсиллиума с помощью РНК In Situ Hybridization (RNA ISH) является первым шагом в изучении функции ORs. РНК ISH использует меченый комплементарный зонд РНК для измерения и локализации специфической последовательности РНК в секции ткани, клеток или целых опор на месте , обеспечивая понимание физиологических процессов и патогенеза болезни. Меченые дигоксигенином (DIG-меченые) и меченные биотином РНК-зонды широко использовались при гибридизации РНК. РНК-маркировка дигоксигенином-11-UTP или биотин-16-UTP может быть получена путем транскрипции in vitro с помощью РНК-полимераз SP6 и T7. DIG- и биотин-меченные РНК-зонды имеют следующие преимущества: нерадиоактивные; безопасно; стабильная; Высокочувствительный; Высокоспециализированный; И легко производить с использованием ПЦР и транскрипции in vitro . DIG- и биотин-меченные РНК-зонды могут быть хромогенными и флуоресцентными. DIG-меченые РНК-зонды могут быть обнаружены с помощью анти-дигоксигенина щелочиNe Phosphatase (AP) -конъюгированные антитела, которые могут быть визуализированы либо с помощью высокочувствительных хемилюминесцентных субстратов нитроилтетразолия хлорида / 5-бром-4-хлор-3-индолилфосфата толуидиновой соли (NBT / BCIP) с использованием оптического микроскопа или с 2 -гидрокси-3-нафтойной кислоты-2&#39;-фениланилидфосфата (HNPP) в сочетании с хлоридом хлорида 4-хлор-2-метилбензолдиазония (Fast Red) с использованием конфокального микроскопа. Меченые биотином РНК-зонды могут быть обнаружены с анти-биотин-стрептавидином конъюгированной пероксидазой (HRP) -конъюгированными антителами, которые можно визуализировать с помощью флуоресцеин-тирамидов с использованием конфокального микроскопа. Таким образом, двухцветная флуоресцентная гибридизация in situ может быть выполнена для обнаружения двух генов-мишеней в одном срезе с использованием меченых DIG- и биотином РНК-зондов. РНК ISH с DIG- и / или биотин-мечеными зондами успешно используется для локализации генов, связанных с обонятельной способностью, таких как OR , ионотропный рецептор, связывающий одорант белокИ мембранного белка сенсорной нейроны, в насекомых-антеннах, но не ограничиваясь ими, Drosophila melanogaster , Anopheles gambiae , L. migratoria и пустынной саранчи Schistocera gregaria 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 . Однако при выполнении РНК ISH для ORS насекомых существуют две существенные проблемы: (1) OR гены (кроме ORco ) выражены на низких уровнях и только в нескольких клетках, что затрудняет обнаружение сигнала и (2) сохранение ткани насекомых для Гистологии, так что морфология сохраняется и фоновый шум является низким, может быть сложным. В этой статье представлен подробный и эффективный протокол, описывающий РНК ISH для локализации генов OR у насекомыхПредставлены антенны, в том числе обнаружение хромогенного и тирамидного сигналов (TSA).

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2&#215;TSINGKETM Master Mix</td>
			<td>TSINGKE, China</td>
			<td>TSE004</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free H2O</td>
			<td>TIANGEN, China</td>
			<td>RT121-02</td>
			<td></td>
		</tr>
		<tr>
			<td>REGULAR AGAROSE G-10</td>
			<td>BIOWEST, SPAIN</td>
			<td>91622</td>
			<td></td>
		</tr>
		<tr>
			<td>Binding buffer</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance buffer</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash solution</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>T Vector</td>
			<td>Promega, USA</td>
			<td>A362A</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega, USA</td>
			<td>M180A</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>TIANGEN, China</td>
			<td>CB101</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma, USA</td>
			<td>A-6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Inalco, USA</td>
			<td>1758-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside</td>
			<td>SBS Genetech, China</td>
			<td>GX1-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nco I</td>
			<td>BioLabs, New England</td>
			<td>R0193S</td>
			<td></td>
		</tr>
		<tr>
			<td>Spe I</td>
			<td>BioLabs, New England</td>
			<td>R0133M</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG RNA Labeling Kit</td>
			<td>Roche, Switzerland</td>
			<td>11175025910</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin RNA Labeling Kit</td>
			<td>Roche, Switzerland</td>
			<td>11685597910</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>DIG RNA Labeling Kit, Roche, Switzerland</td>
			<td>11175025910</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sinopharm, China</td>
			<td>10012718</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm, China</td>
			<td>10009257</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>BEIJING CHEMICAL REGENTS COMPANY, China</td>
			<td>10000292</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura Finetek Europe, Zoeterwoude, Netherlands</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>TINA JIN HAO YANG BIOLOGCAL MANUFACTURE CO., LTE, China</td>
			<td>FISH0010</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sinopharm, China</td>
			<td>80070591</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex</td>
			<td>Millipore, USA</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>AMRESCO, USA</td>
			<td>0777-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitroblue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine salt</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm, China</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>0.04mol/L Tris-Base</td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>BEIJING CHEMICAL REGENTS COMPANY, China</td>
			<td>10000292</td>
			<td>0.12%acetic acid</td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>Sigma, USA</td>
			<td>03677</td>
			<td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>10g/L NaCl</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>MERCK, Germany</td>
			<td>VM335231</td>
			<td>10g/L Peptone from casein (Tryptone)</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>MERCK, Germany</td>
			<td>VM361526</td>
			<td>5g/L Yeast extract</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>10g/L NaCl</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>MERCK, Germany</td>
			<td>VM335231</td>
			<td>10g/L Peptone from casein (Tryptone)</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>MERCK, Germany</td>
			<td>VM361526</td>
			<td>5g/L Yeast extract</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>WISENT ING, Canada</td>
			<td>800-010-CG</td>
			<td>15g/L Agar Bacteriological Grade</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>8.5%NaCl</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sigma, USA</td>
			<td>V900041-500G</td>
			<td>14mM KH2PO4</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sigma, USA</td>
			<td>V900268-500G</td>
			<td>80mM Na2HPO4</td>
		</tr>
		<tr>
			<td>10&#215;Tris buffered saline (pH7.5)</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>1M Tris-Base</td>
		</tr>
		<tr>
			<td>10&#215;Tris buffered saline (pH7.5)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>1.5M NaCl</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>100mM Tris-Base</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>100mM NaCl</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sigma, USA</td>
			<td>V900020-500G</td>
			<td>50mM MgCl2&#183;6H2O</td>
		</tr>
		<tr>
			<td>20&#215;saline-sodium citrate (pH7.0)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>3M NaCl</td>
		</tr>
		<tr>
			<td>20&#215;saline-sodium citrate (pH7.0)</td>
			<td>Sigma, USA</td>
			<td>V900095-500G</td>
			<td>0.3M Na-Citrate</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution (pH9.5)</td>
			<td>Sigma, USA</td>
			<td>V900894-100G</td>
			<td>4% paraformaldehyde</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution (pH9.5)</td>
			<td>Sigma, USA</td>
			<td>V900182-500G</td>
			<td>0.1M NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Buffer (pH10.2)</td>
			<td>Sigma, USA</td>
			<td>V900182-500G</td>
			<td>80mM NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Buffer (pH10.2)</td>
			<td>Sigma, USA</td>
			<td>S7795-500G</td>
			<td>120mM Na2CO3</td>
		</tr>
		<tr>
			<td>Formamide Solution (pH10.2)</td>
			<td>MPBIO, USA</td>
			<td>FORMD002</td>
			<td>50% Deionized Formamide</td>
		</tr>
		<tr>
			<td>Formamide Solution (pH10.2)</td>
			<td></td>
			<td></td>
			<td>5&#215;saline-sodium citrate</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1% Blot</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td>AMRESCO, USA</td>
			<td>0694-500ML</td>
			<td>0.03% Triton X-100</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td></td>
			<td></td>
			<td>1&#215;Tris buffered saline</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase solution</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase solution</td>
			<td></td>
			<td></td>
			<td>Blocking Buffer in Tris buffered saline</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>1% anti-biotin streptavidin horse radish peroxidase- conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td></td>
			<td></td>
			<td>Blocking Buffer in Tris buffered saline</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>MPBIO, USA</td>
			<td>FORMD002</td>
			<td>50% Deionized Formamide</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td></td>
			<td></td>
			<td>2&#215;saline-sodium citrate</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>Sigma, USA</td>
			<td>D8906-50G</td>
			<td>10% dextran sulphate</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>invitrogen, USA</td>
			<td>AM7119</td>
			<td>20 &#181;g/ml yeast t-RNA</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>Sigma, USA</td>
			<td>D3159-10G</td>
			<td>0.2 mg/ml herring sperm DNA</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td>Roche, Switzerland</td>
			<td>11758888001</td>
			<td>1% 2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate (10mg/ml)</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td>Roche, Switzerland</td>
			<td>11758888001</td>
			<td>1% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (25mg/ml)</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td></td>
			<td></td>
			<td>Detection Buffer</td>
		</tr>
		<tr>
			<td>Tyramide signal amplification substrate</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>2% fluorescein-tyramides</td>
		</tr>
		<tr>
			<td>Tyramide signal amplification substrate</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>Amplification Diluent</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Instrument</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Leica, Nussloch, Germany</td>
			<td>Jung CM300 cryostat</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo SCIENTIFIC, USA</td>
			<td>NANODROP 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>Olympus IX71microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>Olympus BX45 confocal microscope</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization of odorant receptor genes in locust antennae by rna in situ hybridization

Насекомые развили сложные системы обонятельного приема, чтобы ощущать экзогенные химические сигналы. Эти химические сигналы трансдуцируются ольфакторными рецепторными нейронами (ORN), расположенными в волоскоподобных структурах, называемых хемосензиллами антенн. На мембранах ORNs, считается, что рецепторы одорантов (ORs) участвуют в кодировании запаха. Таким образом, способность идентифицировать гены, локализованные в ORN, необходима для распознавания OR генов и обеспечивает фундаментальную основу для дальнейших функциональных исследований in situ . Уровни экспрессии РНК специфических OR s в антеннах насекомых очень низки, а сохранение ткани насекомых для гистологии является сложной задачей. Таким образом, трудно локализовать OR на определенный тип сенсиллы, используя гибридизацию РНК in situ. В этой статье вводится подробный и высокоэффективный протокол гибридизации РНК in situ, особенно для слабо выраженных OR генов насекомых. Кроме того, конкретный OR Ген ваИдентифицированных путем проведения двухцветных флуоресцентных экспериментов по гибридизации in situ с использованием ко-экспрессирующего рецепторного гена Orco в качестве маркера.

При хромогенном обнаружении небольшое подмножество антенных клеток в каждой взрослой антенной секции было обозначено DIG-мечеными антисмысловыми зондами LmigOR1 и LmigOR2 ( рис. 3 ). РНК ISH на последовательных участках для локализации LmigOR1 и LmigO...

Watch this Scientific Journal Video about Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization at JoVE.com

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

В этой статье описывается подробный и высокоэффективный протокол гибридизации РНК in situ, в частности, для низкоуровневых экспрессированных генов одорантного рецептора (OR), а также других генов у антенн насекомых с использованием меток дигоксигенина (DIG) или меченых биотином.

Локализация генов-рецепторов одоранта в аутоантителе с помощью РНК<em&gt; Разное</em&gt; Гибридизация

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

The overall goal of this procedure is to localize low level expressed odorant receptor genes as well as other genes in the insect antennae using digoxigenin labeled or biotin labeled probes. To begin this procedure, select the new molting adult locusts that are active and have intact antennae, then cut the antennae into two to three millimeter pieces using sterile razors. Next, apply OCT compound on a freezing microtome holder and place the samples on the compound horizontally.Transfer the holder into the freezing microtome at negative 24 degrees Celsius to equilibrate until the compound freezes. Afterward, take out the holder and cover the samples with a little compound. Transfer the holder into the freezing microtome at negative 24 degrees Celsius again for at least 10 minutes, then section the frozen samples into 12 micrometer thick slices.Subsequently, thaw out the slices one by one on slides and air dry them for 10 minutes. After preparing the cryostat sections, place the slides in a plastic container and fix the tissues by incubating the slides in 4%PFA solution for 30 minutes at four degrees Celsius. After 30 minutes, wash the slides in one X PBS for one minute.To eliminate the alkaline proteins, transfer the slides to 0.2 molar hydrogen chloride for 10 minutes, then transfer the slides to one X PBS with 1%Triton X-100 for two minutes to eliminate the surface protein of nucleic acid. Next, wash the slides twice for 30 seconds in one X PBS. Subsequently, rinse the slides in formamide solution for 10 minutes at four degrees Celsius.Drain the slides and add 100 microliters of diluted antisense and sense probes, then place the cover slips on the tissue sections. Afterward, add formamide solution or one X PBS to the bottom of the box to keep the environment moist, but do not submerge the slides in the liquid. Following this, place the covered slides horizontally in a humid box and incubate at 55 degrees Celsius for 22 hours.After hybridization, remove the cover slips carefully. Wash the sides twice by gently agitating them on a rocker for 30 minutes in 0.1 X SSC at 60 degrees Celsius, then rinse the slides in one X TBS for 30 seconds. Add one milliliter of 1%blocking reagent in TBS supplemented with 0.03%Tritin X-100 on each slide and incubate for 30 minutes.Afterward, discard the blocking solution. For chromogenic detection, use blocking reagent in TBS to dilute 750 units per milliliter anti-digoxigenin-AP conjugated antibody to 1.5 units per milliliter AP solution. Subsequently, add 100 microliters of AP solution to each covered slide.For TSA detection, use blocking reagent in TBS to dilute 750 units per milliliter of anti-digoxigenin-AP conjugated antibody and anti-biotin streptavidin HRP conjugated antibody to the AP HRP solution, then add 100 microliters of AP HRP solution to each covered slide. Add the formamide solution or one X PBS to keep the box moist but not soggy. Following that, incubate the slides in a humid box for 60 minutes at 37 degrees Celsius.Wash the slides three times for five minutes in one X TBS supplemented with 0.05%TWEEN 20 on a rocker. After that, rinse the slides in DAP buffer for five minutes by leaving the plastic container on a rocker agitated gently. For chromogenic detection, add 100 microliters of NBT/BCIP substrate solution to each slide.Carefully place the cover slips onto the slides, then incubate the slides in a humid box with substrate solution for 10 minutes to overnight at 37 degrees Celsius. Check the development by looking at the slides from time to time under the microscope. When the development is ready, stop the reaction by transferring the slides into water.For TSA detection, use a syringe to move the HNPP/Fast Red substrate from the syringe filter, then add 100 microliters of HNPP/Fast Red substrate to each slide covered with a cover slip. After that, incubate the slides for 30 minutes with HNPP/Fast Red substrate at room temperature. After 30 minutes, remove the cover slips carefully and wash the slides three times for five minutes in one X TBS supplemented with 0.05%TWEEN 20.Incubate the slides with 100 microliters of TSA substrate per covered slide for 10 minutes at room temperature. Remove the cover slips carefully and wash the slides three times for five minutes in one X TBS supplemented with 0.05%TWEEN 20 before embedding the slides in PBS glycerol. For chromogenic detection, observe the tissue sections using an optical microscope.Shown here are the labeling pattern of LmigOR1 and LmigORco antisense probe on the consecutive sections of locust antenna. Here are the labeling pattern of LmigORco and LmigOR2 antisense probe on the consecutive sections of locust antenna. The illustration of the occasionally labeled dendritic-like structures are shown here.For TSA detection, observe the tissue sections using a confocal microscope. Here, two color in situ hybridization was performed on the longitudinal antennal section to illustrate the expression of LmigOR1 and LmigORco. Localization of LmigOR1 expressing cells in cell clusters expressing LmigORco confirmed its neural identity, and here is a close view of the boxed areas.Occasionally labeled dendritic-like structure is indicated by an arrow, and the circled areas indicate the olfactory receptor neurons cluster expressing LmigORco and sharing the same sensillum.

Research

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Neuroscience

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Дважды флуоресценции На месте Гибридизация в пресной разделы мозга

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Объединение Липофильные краситель, На месте Гибридизация, иммуногистохимии, и гистологии

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

Multi-единицы Запись методы для характеристики нейронной активности в Locust (<em&gt; Schistocerca Americana</em&gt;) Обонятельные схемы

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Обнаружение генома и стенограммы о стойких вирусов ДНК в нейронных тканей люминесцентными В месте Гибридизация сочетании с Иммуноокрашивание

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Обнаружение Axonally локализованного мРНК в срезах мозга с помощью высоким разрешением<em&gt; На месте</em&gt; Гибридизация

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Сочетание Double Флуорецсенция<em&gt; В Ситу</em&gt; Гибридизация с Immunolabelling для обнаружения экспрессии трех генов в мышином Срезы мозга

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

Жить клеточной измерение одоранта рецептор активации с помощью реального времени лагеря Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Идентификация Дофаминовый рецептор D1-альфа в пределах грызунов прилежащем инновационные РНК в месте обнаружения технологии

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Режиме реального времени в Vitro мониторинга одоранта рецептор активации одоранта в паровой фазе

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Анализ Spliceosomal snRNA Локализация в клетках гели человека с использованием микроинъекции

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...