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

Dette arbeidet støttes av et tilskudd fra National Natural Science Foundation of China (No.31472037). Omtale av handelsnavn eller kommersielle produkter i denne artikkelen er utelukkende for å gi spesifikk informasjon og innebærer ikke anbefaling.

MERK: For å begrense RNA-nedbrytning, lag oppløsninger ved hjelp av vått autoklavert destillert vann (ved 121 ° C i 60 minutter) og også våt-autoklavmaterialer. 1. Fremstilling av RNA ISH Antisense og Sense Probes <ol><li> Målgenforsterkning og rensing <ol><li> Først produserer du et 387 bp dobbeltstrenget fragment av L. migratoria OR1 ( LmigOR1 , GenBank: JQ766965) fra plasmidet som inneholder full lengde-cDNA av LmigOR1 med en Taq</...

<ol>
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F., Wang, G., Su, C. Y., Zwiebel, L. J., 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=Odorant+reception+in+the+malaria+mosquito+Anopheles+gambiae.">Odorant reception in the malaria mosquito Anopheles gambiae.</a> Nature. 464 (7258), 66-71 (2010).</li><li>Hallem, E. A., 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=Coding+of+odors+by+a+receptor+repertoire.">Coding of odors by a receptor repertoire.</a> Cell. 125 (1), 143-160 (2006).</li><li>Wang, G., Carey, A. F., Carlson, J. R., Zwiebel, L. 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>

Det er vanskelig å utføre RNA ISH for å lokalisere OR-gener i insektantenner fordi uttrykksnivåene av OR-gener, med unntak av ORco , er svært lave og bevaring av histologiske skiver av insektantenner er svært vanskelig. I tillegg er TSA-deteksjon også veldig vanskelig. For å løse disse problemene bør følgende tiltak tas. Antennene er utvalgt fra nylig smeltende voksenhoppe som har tynne og myke antennalskiklinger, som opprettholder sin morfologi på lysbildet. De frosne prøvene er delt inn i 12 μm ...

Insektantenner, som er de viktigste kjemosensoriske organene, er dekket av mange hårlignende strukturer - kalt sensilla - som er innervert av Olfactory Receptor Neurons (ORNs). På membranet av insekt-ORN, uttrykkes luktemiddelreceptorer (ORs), en type protein som inneholder syv transmembrane domener, med en coreceptor (ORco) for å danne en heteromer som fungerer som en luktende gated ion-kanal 1 , 2 , 3 . Ulike ORs reagerer på forskjellige kombinasjoner av kjemiske forbindelser 4 , 5 , 6 . Gresshopper ( Locusta migratoria ) stole hovedsakelig på olfaktoriske tegn for å utløse viktige oppføringer 7 . Locust ORs er nøkkelfaktorer for forståelse av molekylære olfaktoriske mekanismer. Lokalisering av et spesifikt locust OR-gen til nevronen av aMorfologisk spesifikk sensillum type ved RNA In situ Hybridization (RNA ISH) er det første trinnet i å utforske ORs-funksjonen. RNA ISH bruker en merket komplementær RNA-probe for å måle og lokalisere en spesifikk RNA-sekvens i seksjon av vev, celler eller hele fester på stedet , som gir innsikt i fysiologiske prosesser og sykdomspatogenese. Digoksigenin-merkede (DIG-merkede) og biotin-merkede RNA-prober har blitt mye brukt i RNA-hybridisering. RNA-merking med digoxigenin-11-UTP eller biotin-16-UTP kan fremstilles ved in vitro transkripsjon med SP6- og T7-RNA-polymeraser. DIG- og biotin-merkede RNA-prober har følgende fordeler: ikke-radioaktive; sikker; stabil; Svært følsom; Svært spesifikk; Og lett å produsere ved hjelp av PCR og in vitro transkripsjon. DIG- og biotin-merkede RNA-prober kan være kromogenisk og påvises fluorescens. DIG-merkede RNA-prober kan påvises med anti-digoksigenin-alkaliNe-fosfatase (AP) -konjugerte antistoffer som kan visualiseres enten med de meget følsomme kjemiluminescerende substratene nitroblue tetrazoliumklorid / 5-brom-4-klor-3-indolylfosfattoluinsalt (NBT / BCIP) ved bruk av et optisk mikroskop eller med 2 -hydroksy-3-naftosyre-2&#39;-fenylanilidfosfat (HNPP) koplet med 4-klor-2-metylbenzenedioniumhemi-zinkkloridsalt (Fast Red) ved bruk av et konfokalt mikroskop. Biotin-merkede RNA-prober kan påvises med anti-biotin streptavidin Hest Radish Peroxidase (HRP) -konjugerte antistoffer som kan visualiseres med fluorescein-tyramider ved hjelp av et konfokalt mikroskop. Dermed kan to-farges fluorescerende in situ- hybridisering utføres for å oppdage to målgener i ett skive ved bruk av DIG- og biotin-merkede RNA-prober. RNA ISH med DIG- og / eller biotin-merkede prober har blitt brukt til å lokalisere olfaktorrelaterte gener, som for eksempel OR , ionotrop reseptor, luktende bindende proteinOg sensorisk nevronmembranprotein, i insektantenner av, men ikke begrenset til, Drosophila melanogaster , Anopheles gambiae , L. migratoria og ørkensprutstenen Schistocera gregaria 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 . Imidlertid er det to betydelige utfordringer når man utfører RNA ISH for insekt-OR: (1) ELLER (unntatt ORco ) uttrykkes i lave nivåer og bare i noen få celler, noe som gjør signaldeteksjon svært vanskelig, og (2) bevare insektvev for Histologi, slik at morfologien blir bevart og bakgrunnsstøyen er lav, kan være utfordrende. I dette papiret er en detaljert og effektiv protokoll som beskriver RNA ISH for lokalisering av OR-gener i insektAntenner presenteres, inkludert både kromogen og tyramid signalforsterkning (TSA) deteksjon.

<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

Insekter har utviklet sofistikerte olfaktoriske mottakssystemer for å fornemme eksogene kjemiske signaler. Disse kjemiske signalene blir transdusert av Olfactory Receptor Neurons (ORNs) plassert i hårlignende strukturer, kalt chemosensilla, fra antennene. På ORN-membranene antas det at odorantreceptorer (OR) er involvert i luktkoding. Det er derfor nødvendig å identifisere gener som er lokalisert til ORNene, for å gjenkjenne OR-gener, og gir et grunnleggende grunnlag for videre funksjonelle in situ- studier. RNA-ekspressionsnivåene for spesifikke OR s i insektantenner er svært lave, og bevaring av insektvev for histologi er utfordrende. Det er således vanskelig å lokalisere en OR til en bestemt type sensilla ved bruk av RNA in situ- hybridisering. I dette papiret blir en detaljert og svært effektiv RNA in situ- hybridiseringsprotokoll spesielt for lavt uttrykte OR-gener av insekter innført. I tillegg er en bestemt OR Gen waS identifisert ved å utføre to-farges fluorescerende in situ- hybridiseringseksperimenter ved bruk av et co-uttrykkende reseptorgen, Orco , som en markør.

Ved kromogen gjenkjenning ble en liten delmengde av antennalcellerne i hver voksenantennseksjon betegnet av de DIG-merkede LmigOR1- og LmigOR2- antisensprober ( Figur 3 ). RNA ISH på påfølgende seksjoner for å lokalisere LmigOR1 og LmigOR2 viste at antennalceller som uttrykker de to gene, befant seg i ORN-klynger som uttrykker LmigORco , hvilket indikerer at den formodede LmigOR1 og 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

Dette papiret beskriver en detaljert og svært effektiv RNA in situ hybridiseringsprotokoll, spesielt for lavnivå uttrykt luktemiddelreceptor (OR) -gener, så vel som andre gener, i insektantenner ved bruk av digoxigenin (DIG) -mærkede eller biotin-merkede prober.

Lokalisering av luktningsmiddelreceptorgener i Locust Antenna av RNA<em&gt; I situ</em&gt; Hybridisering

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

Research

JoVE Journal

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

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

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-enheten opptaket Metoder for å karakterisere nevrale aktiviteten i Locust (<em&gt; Schistocerca Americana</em&gt;) Olfactory kretser

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

Påvisning av Genome og transkripsjoner av en vedvarende DNA virus i nerve vev ved Fluorescent In situ Hybridisering Kombinert med Immunostaining

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

Påvisning av Axonally Lokalisert mRNAs i hjernedelene med høy oppløsning<em&gt; In Situ</em&gt; Hybridisering

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

Kombinere Double Fluorescence<em&gt; In Situ</em&gt; Hybridisering med immunomerking for Påvisning av Expression of Three Gener i musehjerne §§

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

Live-celle måling av Odorant reseptor aktivisering benytter en sanntid leiren analysen

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

Identifikasjon av dopamin D1-Alpha reseptor i gnager Nucleus Accumbens av en nyskapende RNA i Situ oppdagelsen teknologien

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

Sanntid i Vitro overvåking av Odorant reseptor aktivering av en Odorant i damp fase

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

Analyse av Spliceosomal snRNA lokalisering i Human Hela Cells bruke Microinjection

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