Name: depletion of ribosomal rna for mosquito gut metagenomic rna-seq
Uploaded: 2013-04-07T14:00:00
Description: Watch this Scientific Journal Video about Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq at JoVE.com

Dette arbejde blev støttet af NIH tilskud 1SC2GM092789-01A1, og MS var et forsknings lærd af NMSU Howard Hughes Medical Institution Undergraduate forskningsprogrammer. Videoen blev instrueret og produceret af Amy Lanasa og koordineret af Dr. Philip Lewis med Creative Media Institut på NMSU.

Procedure 1. Mosquito Opdræt <ol><li> Bageste myg Anopheles gambiae G3-stamme i en insectary ved 27,5 ° C med 80% fugtighed og 12:12 hr cyklus af lys / mørke. </li><li> Foder larver med jorden kattefoder med ølgær i forholdet 1:1. </li><li> Feed voksne myg på mus blod på dag 3 efter fremspiring til ægproduktion. </li></ol> 2. Mosquito Gut Dissection <ol><li> Autoklave Dissektionsværktøj (dias og pincet). </li><li> Saml 50 myg ved hjælp af en aspir...

<ol>
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En kompleks mikrobielle samfund ligger i den myg tarmen økosystem 14,16,17. Metatranscriptomic sekventering (RNA-seq) kan afsløre kontekstafhængige funktionel information ved at udspørge hele mikrobielle transkriptomet 4,18. Teknisk, oligo-d (T) medieret berigelse af prokaryotisk mRNA er ikke anvendelig på grund af fraværet af stabile poly (A) haler budbringere. Alternativt er rRNA depletion blevet anvendt til mRNA berigelse. Her har vi udviklet en subtraktion-baseret protokol til nedbryder r...

Næste generation sekventering teknologi har i høj grad avanceret metagenomet undersøgelse ved at tillade at vurdere taksonomiske sammensætning og genetisk funktionalitet af en mikrobiel assemblage. RNA-sekventering (RNA-Seq) 1 kan omgå kultur-baserede metoder til at undersøge mikrobielle metatranscriptomes i forskellige sammenhænge 2-5. En stor hindring for mikrobiel RNA-seq er vanskeligheden ved at berige mRNA, som de prokaryote mRNA-species der ikke er stabilt polyadenyleret. Derfor oligo d (T) medieret messenger berigelse finder ikke anvendelse. Fjernelse af rigelige rRNA er en alternativ fremgangsmåde til at berige mRNA. Kommercielle rRNA depletion kits, såsom Microexpress Bakteriel mRNA Enrichment kit (Ambion), RiboMinus transkriptom Isolation Kit (bakterier) (Life Technologies), og mRNA-ONLY Prokaryotisk mRNA Isolation kit (Epicentre), som fortrinsvis nedbrydes rRNA med en exonuclease, er blevet anvendt til fjernelse af rRNA 6-8. Men bindingsproberne i Microexpresseller RiboMinus er gode til at fjerne kendte rRNA fra typiske Gram-positive og Gram-negative bakterier (se fabrikantens specifikationer), men mindre kompatibel med rRNA fra ukendte mikrober. Derfor kan fjernelse være mindre effektiv for metagenomic prøver 8-10. Desuden mRNA overflod fidelity var tvivlsom, da exonukleasebehandling blev anvendt 11. Samlet subtraktion-baserede rRNA depletion var mindre forudindtaget og mere effektive i mRNA berigelse i metagenomic indstillinger 10-13. Den myg gut rummer en dynamisk mikrobielle samfund 14. Vi er interesseret i at karakterisere funktionen af ​​den myg tarmen microbiome ved hjælp af RNA-Seq. I en RNA-prøve isoleret fra de myg indvolde, er begge myg og mikrobielle RNA til stede. Her beskriver vi en modificeret protokol til at bruge community specifikke rRNA prober til effektivt nedbryder myg og mikrobiel rRNA ved subtraktiv hybridisering. Den resulterende mRNAberigede prøver er egnede til RNA-Seq. Den samlede arbejdsgang er afbildet i figur 1..

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material </td>
 </tr>
 <tr>
 <td>Meta-G-Nome 				DNA isolation kit</td>
 <td>Epicentre</td>
 <td>MGN0910</td>
 <td>Metagenomic 				DNA isolation</td>
 </tr>
 <tr>
 <td>TriPure </td>
 <td>Roche</td>
 <td>11667165001</td>
 <td>Mdetagenomic 				RNA isolation</td>
 </tr>
 <tr>
 <td>MEGAscript 				T7 kit</td>
 <td>Ambion</td>
 <td>AM1334</td>
 <td>In 				vitro synthesis of RNA probes</td>
 </tr>
 <tr>
 <td>RNaseZap</td>
 <td>Ambion</td>
 <td>AM9780</td>
 <td>RNase 				free working area </td>
 </tr>
 <tr>
 <td>Biotin-16-UTP</td>
 <td>Roche</td>
 <td>11388908910</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Biotin-11-CTP</td>
 <td>Roche</td>
 <td>4739205001</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Streptavidin 				magnetic beads</td>
 <td>NEB</td>
 <td>S1420S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>Magnetic 				separation rack</td>
 <td>NEB</td>
 <td>S1506S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>RNeasy 				mini kit</td>
 <td>QIAGEN</td>
 <td>74104</td>
 <td>Purification 				of subtracted RNA</td>
 </tr>
 <tr>
 <td>RNase-Free 				DNase Set</td>
 <td>QIAGEN</td>
 <td>79254</td>
 <td>Removal 				DNA contamination </td>
 </tr>
 <tr>
 <td>Agilent 				RNA 6000 Pico Kit</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>5067-1513</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Bio-Gen 				PRO200 				Homogenizer</td>
 <td>PRO 				Scientific</td>
 <td>01-01200</td>
 <td>Mosquito 				gut tissue homogenization</td>
 </tr>
 <tr>
 <td>NanoDrop 				1000 Spectrophotometer</td>
 <td>Thermo 				Scientific</td>
 <td></td>
 <td>DNA 				&amp; RNA quantitation</td>
 </tr>
 <tr>
 <td>2100 				Bioanalyzer</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>G2940CA</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 </tbody>
</table>

depletion of ribosomal rna for mosquito gut metagenomic rna-seq

Den myg gut rumme dynamiske mikrobielle samfund på tværs af forskellige stadier af insektets livscyklus. Karakterisering af den genetiske kapacitet og funktionalitet af tarmen samfund vil give indsigt i følgerne af tarmens mikrobiota om myg liv træk. Metagenomic RNA-Seq er blevet et vigtigt værktøj til at analysere transcriptomes fra forskellige mikrober til stede i en mikrobiel samfund. Messenger RNA omfatter sædvanligvis kun 1-3% af totalt RNA, mens rRNA udgør cirka 90%. Det er en udfordring at berige messenger-RNA fra en metagenomic mikrobiel RNA-prøve, fordi de fleste prokaryote mRNA-arter mangler stabile poly (A) haler. Dette forhindrer oligo d (T) medieret mRNA isolation. Her beskriver vi en protokol der anvender prøve afledt rRNA indfangningsprober til fjernelse af rRNA fra en metagenomic total RNA-prøve. Til at begynde, er både myg og mikrobielle små og store subunit rRNA-fragmenter amplificeret fra et metagenomic samfund DNA-prøve. Derefter kommunikatiskabets specifikke biotinylerede antisense ribosomale RNA-prober syntetiseres in vitro under anvendelse af T7 RNA-polymerase. De biotinylerede rRNA-prober hybridiseret til totalt RNA. Hybriderne indfanges af streptavidin-coatede beads og fjernes fra det totale RNA. Denne subtraktion-baseret protokol effektivt fjerner både myg og mikrobiel rRNA fra den totale RNA-prøve. MRNA&#39;et beriget prøve yderligere forarbejdes for RNA-amplifikation og RNA-Seq.

Protokollen indeholder tre afsnit: (1) udarbejdelse af metagenomic DNA skabeloner til rRNA PCR, (2) skabelse af prøve specifikke capture rRNA prober, (3) udtømning af rRNA fra total RNA ved subtraktiv hybridisering. Isolering af høj kvalitet metagenomic DNA og RNA er afgørende for hele processen. Det modificerede Meta-G-Nome DNA-isolering protokol giver høj kvalitet metagenomic DNA fra myg indvolde, som vist i figur 2. Det kan være udfordrende at isolere høj kvalitet total RNA fra myg indvolde. D...

Watch this Scientific Journal Video about Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq at JoVE.com

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

En ribosomalt RNA (rRNA) udtømning protokol blev udviklet til at berige messenger RNA (mRNA) for RNA-seq af myg tarmen metatranscriptome. Prøve specifikke rRNA prober, som blev brugt til at fjerne rRNA via subtraktion, blev skabt fra myg og dens tarm mikrober. Udførelsen af ​​protokollen kan resultere i fjernelse af tilnærmelsesvis 90-99% af rRNA.

Udtømning af ribosomalt RNA for Mosquito Gut Metagenomic RNA-seq

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