Bu çalışma NIH hibe 1SC2GM092789-01A1 tarafından desteklenen, ve MS NMSU Howard Hughes Tıp Enstitüsü Lisans Araştırma Programları bir araştırma bilim adamı oldu. Video yönetmen ve yapımcı Amy Lanasa tarafından NMSU de Creative Media Enstitüsü ile Dr Philip Lewis tarafından koordine edilmiştir.

Prosedür 1. Sivrisinek Yetiştirme <ol><li> 27.5 bir insectary Arka sivrisinek Anofel gambiae G3 zorlanma ° C / karanlık ışığın% 80 nem ve 12:12 saat döngüsü ile. </li><li> Oranı 1:1 bira mayası ile zemin kedi maması ile larva besleme. </li><li> 3. gün yumurta üretimi için post-ortaya Farelerde kan yetişkin sivrisinekler besleyin. </li></ol> 2. Sivrisinek Gut Diseksiyon <ol><li> Otoklav diseksiyon araçları (slaytlar ve forseps). </li><li> Bir aspiratörü kullanarak 50 sivrisinek toplayın ve CO 2 akış yatağı (Ultimate Flypad) üzerine koyun. Sivrisinek vücut yüzeyi temizlemek için% 70 etanol içeren üç petri sırayla bir sivrisinek örneği durulayın. </li><li> Bir stereomikroskobi altında bir cam slayt üzerine numune yerleştirin. Gut çıkarın. </li><li> Metagenomic DNA izolasyonu için koşul başına 50 cesareti toplayın. Metagenomic RNA izolasyonu için koşul başına 50 cesareti toplayın. </li></ol&gt; 3. Metagenomic DNA İzolasyonu Not: Metagenomic DNA aşağıda açıklanan değişiklik ile Meta-G-Nome DNA izolasyon kiti (Epicentre Biotechnologies) kullanılarak izole edilir. <ol><li> 300 ul TE 50 sivrisinek bağırsaklar yerleştirin. Buz üzerinde hız 2.000 rpm Bio-Gen PRO200 homojenizatör (PRO Bilimsel Inc, ABD) kullanılarak 1 dk için onları homojenize. </li><li> Hazır-Lyse Lizozim Çözüm 2 ul RNaz hücre süspansiyonu A 1 ul ekleyin. 30 dakika 37 ° C&#39;de vorteks ve inkübe Mix by. </li><li> Meta-Lizis çözeltisi (2X) ve tüp ile Proteinaz K ve 1 ul 300 ul ekle. Vorteks ile karıştırın. Kısaca çözeltinin her tüpün tabanında olduğundan emin olmak için tüp darbe santrifüjden, ve 3-5 dakika için buz üzerinde daha sonra oda sıcaklığına kadar soğutulur 15 dak, yer için 65 ° C sıcaklıkta inkübe edilir. </li><li> 10 saniye boyunca kuvvetlice vorteks tüpü ve karıştırın MPC Protein yağış reaktifi 350 ul ekleyin. </li><li> Pelet t4 azından 12,000 x g&#39;de 10 dakika boyunca santrifüj ile çöpleri ° C. </li><li> Temiz bir 2 ml tüp için supernatant aktarın ve pelet atın. </li><li> Süpernatant ile izopropanol 570 ul ekle. Ters tüp birkaç kez karıştırın. </li><li> Pelet 4 12,000 x g&#39;da 10 dakika için santrifüjleme ile DNA ° C. DNA pelet oradan çıkarmadan izopropanol çıkarın. </li><li> Pelet yıkamak için% 75 etanol 500 ul ekleyin. 4 ° C&#39;de 12,000 x g&#39;de 5 dakika boyunca santrifüje DNA pelet bozmadan etanol çıkarın. 2 dakika için oda sıcaklığında pelet Hava kurutun. Not: DNA pelet aşırı kuru etmeyin. </li><li> TE tamponu 50 ul DNA pelet yeniden süspanse edin. </li><li> Bir% 1 agaroz jeli üzerinde jel elektroforezi yolu ile, kitte sağlanan; Fosmid Kontrol DNA (100 ng / ml &#39;den 40 kb) ile kıyasla izole edilmiş DNA&#39;nın boyutu ile doğrula. </li></ol> 4. Toplam RNA İzolasyonu Not: çalışma alanı temizleyinRNaz kontaminasyonu en aza indirmek için RNaseZap ile. Metagenomic RNA, küçük bir değişiklik ile TriPure İzolasyon Reaktif (Roche) kullanılarak sivrisinek gut örneklerden izole edilmiştir. <ol><li> 500 ul TriPure İzolasyon Reaktifi ile 2 ml tüp içinde 50 sivrisinek cesaretlenin. Bir homojenizer kullanarak, buz üzerinde hızlı 2.000 rpm&#39;de 1 dakika için homojenize edilir. Nükleoprotein komplekslerin dissosiyasyon imkan vermek üzere 5 dakika oda sıcaklığında bekletin. </li><li> 12 sn için 100 ul kloroform ve vorteks ekleyin ve 2 dakika boyunca oda sıcaklığında bekletin. </li><li> 10 dakika boyunca 10,000 x g&#39;de santrifüjleyin. Dikkatlice rahatsız ayrılmış aşamaları olmadan tüpü çıkarmak. </li><li> Yeni bir tüp ile sulu faz transfer, 250 ul izopropanol eklemek iyice karıştırılır, ve 20 dakika süre ile -20 ° C&#39;de koydu. </li><li> 4, 10 dakika için 12,000 x g&#39;de santrifüjleyin ° C&#39;de çökelti RNA için. </li><li> Pelet oradan çıkarmadan Süpernatantı atın. 750 ul% 75 etanol ile pelet yıkayın. </li><li> Pelet bozmadan süpernatant kapalı pipet. Air 2 dakika süreyle tüp kurulayın. </li><li> 30 ul nükleaz ücretsiz su ile RNA tekrar. </li><li> 20 dk için 37 ° C &#39;de DNaz ile tedavi edilen toplam RNA ve daha sonra 75 ile inkübe ederek DNase inaktive 15 dakika için ° C. 30 ul nükleaz ücretsiz su etanol ve tekrar süspansiyon RNA ile Çökelti RNA. </li><li> Bir Nanodrop kullanılarak toplam RNA miktarını belirleyin. </li></ol> 5. Ribozomal RNA tükenmesi Not: Bu protokol daha önce tarif edilen yöntemler 12,13,15 üzerinde geliştirilmiştir. <ol><li> Ribozomal RNA gen fragmanlarının PCR amplifikasyon Bu adım, toplam RNA örnek rRNA için ücretsiz olan anti-sense RNA (arRNA) probları üretmek için in vitro transkripsiyon için şablon olarak kullanılacak örnek özgü rRNA havuzları, oluşturur. <ol><li> RRNA fragmanı PCR (Tablo 1) primer setleri tasarlayın ve sentez. </li><li> 50 PCR Çalıştır50 ng DNA şablonu, 1 x PCR tamponu, 1.5mM Mg 2 ile Taq DNA polimeraz (Qiagen) kullanılarak ul reaksiyon, Cl, denaturasyon, 94 ve 35 döngü ile 0.2 uM primerler ° C&#39;de 10 saniye, 40 saniye, 15 ° C&#39;de tavlama için bakteri 23S amplikon, ve 50 ° C diğer amplikonlar ve 72 uzatılması için 1 dk (72 nihai uzatma ° C de 5 dakika) için ° C. </li><li> % 1 agaroz jel üzerinde PCR ürünleri görün. </li><li> 50 ul yıkama tamponu ile elüsyon QIAquick PCR saflaştırma kiti kullanılarak PCR ürünleri temizler. Nanodrop kullanılarak konsantrasyon ölçülmesine imkan verirler. </li></ol></li><li> Biotin-etiketli anti-sense rRNA probları (arRNA) in vitro transkripsiyon olarak. Not: Bu basamakta, çeşitli rRNA gelen amplikonlar örnek özel problar MEGAscript T7 kiti kullanılarak sentezlenmiştir. <ol><li> Aşağıdaki maddeleri (Tablo 2) karıştırılması ile her bir örnek için özel rRNA amplikon bir 20 ul reaksiyon ayarlayın. 37 gece inkübe ° C <strong&gt; Not: genellikle reaksiyon verimi&gt; 50 mg RNA. </li><li> DNA şablonu kaldırmak için 20 dakika boyunca 37 ° C de inkübe etmek ve reaksiyon 1 ul DNase I (kitinde) ekleyin. </li><li> 4, 10 dakika için 12,000 x g&#39;da reaksiyon için 100 ul% 100 etanol, santrifüj ekleme ° C arRNA sentezlendi çökeltilir. Süpernatantı atın ve 750 ul% 75 etanol ile pelet yıkayın. 50 ul nükleaz ücretsiz su süspanse RNA problar. </li><li> Bir Nanodrop kullanarak RNA konsantrasyonu niceliğini. -80 Mağaza probları ° C </li></ol></li><li> biotinlenmiş arRNA ile rRNA çıkarma. Not: Bu adım, toplam RNA örnek rRNA için melezleşme biotinlenmiş arRNA kullanır. RRNA arRNA melezler streptavidin kaplı manyetik boncuk tarafından yakalanır. Prosedür küçük değişiklik ile referans 13&#39;de tarif edilen protokole dayanmaktadır. <ol><li> Reaktifler <ol><li> 0.1 N NaOH, 20X sodyum klor hazırlayınide-sitrat (SSC) ve% 20 formamid 1 X SSC. </li></ol></li><li> Melezleme <ol><li> Bakteriyel 16S ve 23S probları (0.75 mikrogram her), sivrisinek 18S ve 28S probları (0.75 mikrogram adet) 1 mg total RNA karıştırın, 1 ul RNaz inhibitörü, 2.5 ul 20X SSC tamponu, 200 ul PCR tüp 10 ul% 100 formamid , 50 ul ekleyin nükleaz ücretsiz su. </li><li> 70, bir termal döngü cihazı ve inkübe reaksiyon tüpü yerleştirin ° C de 5 dakika ikincil yapı açmak ve 25 aşağı rampa ° C rRNA ve arRNA hibridizasyon probları izin vermek için 1 dakika her biri ° C de 5 ° C lik artışlarla kullanarak. </li></ol></li><li> RRNA Kaldırma <ol><li> 1.7 ml tüp içine 300 ul streptavidin kaplı boncuklar aktarın. Boncuk hareketsiz bir manyetik ayırma raf tüp yerleştirin. Süpernatantı. 0.1N NaOH ve mil ve 300 ul içinde süspanse boncukiyi x. Manyetik raf geri tüpü yerleştirin ve süpernatant kapalı Pipeti. Iyice karıştırın, 1X SSC 300 ul boncuklar tekrar süspansiyon, boncuk immobilize ve süpernatant kapalı Pipeti. 1X SSC bir kez daha 300 ul yıkama tekrarlayın. </li><li> Boncuk 3 alikotları, 100 ul, her biri 1.7 ml tüpler olun. Manyetik raf tüp yerleştirin ve pipetleme tarafından süpernatant kaldırmak. Not: boncuk alikotları biyotinile rRNA arRNA melez yakalama 3 tur için kullanılır. </li><li> 50 ul hibridizasyon reaksiyona girerek% 20 formamid 50 ul 1X SSC ekleyin. İlk boncuk kısım tüpüne reaksiyon aktarın ve iyice karıştırın. Biotinlenmiş prob-rRNA melezleri streptavidin boncuk tarafından yakalanan izin vermek için 10 dakika süreyle oda sıcaklığında inkübe edin. Tüp inkübasyon sırasında karıştırmak için her 2 dakikada Flick. </li><li> Boncuk hareketsiz manyetik raf tüp yerleştirin, ikinci aliq için süpernatant aktarmakboncuk uot, iyice karıştırın ve yukarıdaki gibi kuluçkaya yatmaktadır. Boncuk üçüncü tüp içine Süpernatant aktararak üçüncü yakalama gerçekleştirilmesi. Karıştırın ve inkübe. </li><li> Üreticinin kılavuzunu takip formamid kaldırmak RNeasy MinElute kitini kullanarak yeni bir tüp ve davranış arıtma içine non-rRNA süpernatant aktarın. </li><li> Bir Bioanalyzer (Şekil 4) bir Agilent RNA 6000 Pico yonga seti kullanarak rRNA tükenmesi verimliliğini kontrol edin. </li></ol></li></ol></li></ol> Not: rRNA tüketilmiş RNA numuneleri 8 gerekirse amplifikasyon mRNA tabi tutulur.

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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Karmaşık bir mikrobiyal topluluk sivrisinek bağırsak ekosistemi 14,16,17 bulunur. Metatranscriptomic sıralama (RNA-seq) tüm mikrobiyal transcriptome 4,18 sorgulayarak bağlam bağımlı işlevsel bilgileri açığa çıkartabilir. Teknik olarak, prokaryotik mRNA oligo-d (T) aracılı zenginleştirme dolayı stabil poli haberciler (A) kuyrukları olmaması için geçerli değildir. Alternatif olarak, rRNA mRNA zenginleştirme tabakasının incelmesi için kullanılmıştır. Burada, sivrisinek...

Gelecek nesil dizileme teknolojisi büyük ölçüde taksonomik kompozisyonu ve mikrobiyal topluluk genetik işlevselliğini değerlendirmek için izin vererek metagenomik çalışmada ilerlemiştir. RNA-Dizileme (RNA-Seq) 1 farklı bağlamlarda 2-5 mikrobiyal metatranscriptomes araştırmak için kültüre dayalı yöntemler atlayabilir. Prokaryotik mRNA türleri stabil polyadenylated değil gibi mikrobik RNA-seq için büyük bir engel, mRNA zenginleştirici güçlüktür. Bu nedenle, oligo d (T) aracılı messenger zenginleşme geçerli değildir. Bol rRNA kaldırılmasını mRNA zenginleştirmek için alternatif bir yaklaşımdır. Böyle Microexpress Bakteriyel mRNA Zenginleştirme kiti (Ambion), RiboMinus transcriptome İzolasyon Kiti (Bakteri) (Life Technologies), ve tercihen bir eksonükleaz ile rRNA alçaltır mRNA-SADECE Prokaryotik mRNA İzolasyon kiti (Epicentre) gibi Ticari rRNA tükenmesi kitleri, kullanılmış rRNA 6-8 kaldırmak için. Microexpress Ancak, yakalama problarıya RiboMinus (imalatçılarının şartnamelerine bakın) tipik Gram-pozitif ve Gram-negatif bakteriler bilinen rRNA kaldırılması için iyi, ama bilinmeyen mikroplardan rRNA ile daha az uyumludur. Sonuç olarak, kaldırma Metagenomic örnekler 8-10 için daha az etkili olabilir. Ekzonükleaz tedavi 11 verildiğinde yanı sıra, mRNA bolluk aslına uygunluk sorgulanabilir. Genel olarak, çıkarma tabanlı rRNA tükenmesi az önyargılı ve Metagenomic ayarları 10-13 mRNA zenginleştirme daha etkili olmuştur. Sivrisinek gut dinamik mikrobiyal topluluk 14 barındırmaktadır. Biz RNA-Seq kullanarak sivrisinek bağırsak mikrobiyomları fonksiyonunu karakterize ilgilendi. Sivrisinek bağırsaklar izole edilen bir RNA örnek olarak, her ikisi de mikrobiyal sivrisinek ve RNA bulunmaktadır. Burada, biz verimli eksiltici hibridizasyon ile sivrisinek ve mikrobiyal rRNA tüketmek topluluk belirli rRNA probları kullanmak üzere değiştirilmiş bir protokol açıklar. Elde edilen mRNAzenginleştirilmiş örnekleri RNA-Seq için uygundur. Genel akışı Şekil 1 &#39;de tasvir edilmiştir.

<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

Sivrisinek gut böceğin yaşam döngüsünün farklı aşamaları boyunca dinamik mikrobiyal topluluklar barındırmaktadır. Gut toplumun genetik kapasite ve işlevselliğe Karakterizasyonu sivrisinek hayatı özellikleri üzerine gut mikrobiyotasına etkileri içgörü sağlayacaktır. Metagenomic RNA-Seq bir mikrobiyal topluluk bulunan çeşitli mikroplardan transcriptomes analiz için önemli bir araç haline gelmiştir. RRNA yaklaşık% 90&#39;ını oluşturmaktadır ise mesajcı RNA genellikle total RNA sadece% 1-3 oluşmaktadır. Çoğu prokaryotik mRNA türleri istikrarlı poli (A) kuyrukları eksikliği nedeniyle bir Metagenomic mikrobiyal RNA örnek mesajcı RNA zenginleştirmek için zordur. Bu oligo d (T) aracılı mRNA izolasyon önler. Burada, rRNA bir Metagenomic toplam RNA örnek rRNA kaldırmak için problar yakalamak türetilmiş örnek kullanan bir protokol açıklar. Başlamak için, sivrisinek ve mikrobiyal küçük ve büyük alt ünite rRNA parçaları hem Metagenomic topluluk DNA örneğinden güçlendirilir. Daha sonra, CommuSon veriler özel biyotinli antisense RNA probları T7 RNA polimerazı kullanılarak in vitro olarak sentezlenir. Biotinlenmiş rRNA probları toplam RNA melezi. Melez streptavidin kaplı boncuklar tarafından yakalanır ve toplam RNA kaldırılır. Bu çıkarma tabanlı protokol verimli total RNA örnek sivrisinek ve mikrobiyal rRNA hem kaldırır. MRNA, zenginleştirilmiş bir numune, RNA amplifikasyonu ve RNA-Seq için işleme tabi tutulur.

, (2) numune belirli bir yakalama rRNA probları yaratılması; rRNA (3) azalması eksiltme hibridizasyon ile total RNA rRNA PCR Metagenomic DNA şablonları (1) Hazırlık: Protokol üç bölümden oluşmaktadır. Yüksek kalite Metagenomic DNA ve RNA İzolasyonu tüm süreci esastır. Değiştirilmiş Meta-G-Nome DNA izolasyon protokol olarak Şekil 2&#39;de gösterildiği gibi sivrisinek bağırsaklar yüksek kaliteli Metagenomic DNA, elde edilir. Sivrisinek cesareti yüksek kalitede toplam RNA izole...

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

A ribozomal RNA (rRNA) azalması protokolü sivrisinek bağırsak metatranscriptome RNA-seq mesajcı RNA (mRNA) zenginleştirmek için geliştirilmiştir. Çıkarma yoluyla rRNA kaldırmak için kullanılan örnek belirli rRNA probları, sivrisinek ve onun bağırsak mikropları oluşturulmuş. Protokol performans rRNA yaklaşık olarak% 90-99 arasında kaldırma neden olabilir.

Sivrisinek Gut Metagenomic RNA-seq Ribozomal RNA tükenmesi

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

depletion-of-ribosomal-rna-for-mosquito-gut-metagenomic-rna-seq

Research

JoVE Journal

Biology

19.5K Görüntüleme.  New Mexico State University. A ribozomal RNA (rRNA) azalması protokolü sivrisinek bağırsak metatranscriptome RNA-seq mesajcı RNA (mRNA) zenginleştirmek için geliştirilmiştir. Çıkarma yoluyla rRNA kaldırmak için kullanılan örnek belirli rRNA probları, sivrisinek ve onun bağırsak mikropları oluşturulmuş. Protokol performans rRNA yaklaşık olarak% 90-99 arasında kaldırma neden olabilir.

Video: Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic Kütüphaneler Büyük Ölçekli Ekranlar

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Biyoloji

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Sivrisinek Yetiştirme Protokolü (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Yüksek Verimli Ekran Metagenomic Kütüphaneler selülaz Aktivitesi Biomining

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa Hücre Transfeksiyon Ardından Gaussia Lusiferaz mRNA in vitro transkripsiyon ve Kapatma yılında

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / Cas Sistemleri Endonükleazlar için Substrat Üretimi

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Trombin-tedavi içinde Transcriptomes Analizi ve Kontrol İnsan Pulmoner mikrovasküler endotel hücrelerinde

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

16S Ribozomal RNA Gen amplikonlarının nesil Sıralama

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

Asya Tiger Sivrisinek içinde Photoperiodic Diapause RNA-seq Analizler Deneysel ve Biyoinformatik Protokolü,<em&gt; Aedes albopictus</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Fizyolojik Kayıtlar ve Sarı-ateş Sivrisinek Tat Çıkıntılara RNA Sıralama<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Hastalık Vektör Sivrisinek Larva içinde Kitosan / Müdahale RNA Nanopartikül Aracılı gen susturma

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...