この作品は、NIHの助成金1SC2GM092789-01A1でサポートされており、MSはNMSUハワードヒューズ医学研究所学部研究プログラムの研究学者だった。ビデオは監督と生産エイミーLanasaによってとNMSUのクリエイティブメディア研究所で博士フィリップ·ルイスによってコーディネートされた。

手順 1。蚊飼育<ol><li> 27.5での昆虫館でリア蚊ハマダラカ G3の歪℃の明/暗の湿度80％と12時12時間の周期を持つ。 </li><li>比1:1でビール酵母でグランドキャットフードと幼虫を養う。 </li><li>産卵のために3日目発芽後にマウスの血液を蚊の成虫を養う。 </li></ol> 2。蚊腸解剖<ol><li>オートクレーブ解剖ツール（スライドや鉗子）。 </li><li>アスピレーターを用いて50蚊を収集し、CO 2流量ベッド（アルティメットFlypad）の上に置きます。蚊の体表面をきれいにする70％エタノールを含む3ペトリ皿に順次蚊の標本をすすぐ。 </li><li>実体顕微鏡下のスライドガラス上に試料を置きます。腸を取り出します。 </li><li>メタゲノムDNA単離するための条件あたり50根性を収集します。メタゲノムRNA単離のための条件あたり50根性を収集します。 </li></ol&gt; 3。メタゲノムDNAの単離注：メタゲノムDNAは後述の修正を加えてメタG - ノームのDNA単離キット（エピセンターバイオテクノロジーズ）を用いて単離される。 <ol><li> 300μlのTEで50蚊根性を置きます。氷の上で速度2,000 rpmでバイオジェンPRO200ホモジナイザー（PROの科学社、米国）を用いて、1分のためにそれらをホモジナイズする。 </li><li>リゾチーム溶液と細胞懸濁液へのRNase Aを1μl既製溶解液2μlを追加します。 30分間37℃でインキュベートし、ボルテックスで混和。 </li><li>メタのLysis Solution（2X）とチューブにプロテイナーゼKを1μlの300μlを添加する。ボルテックスで混ぜる。簡単に言うと、ソリューションのすべてをチューブの底にあることを確実にするためにチューブをパルス遠心し、3〜5分間氷上で、次いで室温に15分間で、場所を65℃でインキュベートする。 </li><li> 10秒間激しくボルテックスすることによりチューブとのミックスに、MPCのタンパク質沈殿試薬350μlを添加する。 </li><li>ペレットトン4時に彼12,000 xgで10分間の遠心分離により破片℃、 </li><li>クリーン2 mlチューブに上清を移し、ペレットを捨てる。 </li><li>上清にイソプロパノール570μlを添加する。反転チューブを数回転倒混和する。 </li><li>ペレット4で12,000 xgで10分間遠心分離によりDNA℃、 DNAペレットを外れずにイソプロパノールを削除します。 </li><li>ペレットを洗浄する75％エタノール500μlを添加する。 4℃、12,000 xgで5分間遠心DNAペレットを乱さずにエタノールを除去します。 2分間室温でペレットを空気乾燥。注：DNAペレット過乾燥させないでください。 </li><li> TEバッファー50μlのDNAペレットを再懸濁します。 </li><li> 1％アガロースゲル上で電気泳動を経由して、キットに付属;フォスミドコントロールDNA（100 ng /μlに40キロバイト）と比較することにより単離されたDNAのサイズを検証します。 </li></ol> 4。 total RNA抽出注：作業領域をきれいにRNaseの混入を最小限に抑えるためRNaseZap持つ。メタゲノムRNAはマイナーな修正とTriPure分離試薬（Roche）を使用して蚊の腸の検体から分離された。 <ol><li> 500μlのTriPureアイソレーション試薬による2 mlチューブで50蚊根性を入れてください。ホモジナイザーを用いて氷の上で速度2,000 rpmで1分間ホモジナイズする。核タンパク質複合体の解離を許可するように室温で5分間放置します。 </li><li> 12秒のために、100μlのクロロホルムと渦を追加し、2分間室温で放置します。 </li><li> 10分間、10,000×gで遠心分離します。慎重に、相を分離を乱すことなく、チューブを取り出します。 </li><li>新しいチューブに水相を移し、250μlのイソプロパノールを加えよく混ぜ、20分間-20℃で置く。 </li><li> 4℃で10分間12,000 xgで遠心し沈殿℃のRNAに。 </li><li>ペレットを外れずに、上清を捨てる。 750μlの75％エタノールでペレットを洗浄します。 </li><li>ペレットを乱すことなく、上清をピペットで。 Airは2分間、チューブを乾燥させてください。 </li><li> 30μlのヌクレアーゼフリー水を用いてRNAを再懸濁します。 </li><li> 37℃でDNaseで全RNAを扱う20分間C、およびその後15分間75℃でインキュベートすることにより、DNaseを不活性化する。 30μlのヌクレアーゼフリー水でエタノールに再懸濁しRNAとRNAを沈殿させる。 </li><li>光度計を用いてトータルRNAの量を決定します。 </li></ol> 5。リボソームRNAの枯渇 注：プロトコルは前述の方法12,13,15に基づいて開発されました。 <ol><li>リボソームRNA遺伝子断片のPCR増幅 このステップでは、トータルRNAサンプルのrRNAに相補的であるアンチセンスリボソームRNA（arRNA）プローブを生成するためにin vitro転写用のテンプレートとして使用されるサンプル固有のrRNAのプールを作成します。 <ol><li> rRNAの断片のPCR（ 表1）のプライマーセットを設計し、合成する。 </li><li> 50でPCRを実行する50 ngのDNAテンプレート、1×PCR緩衝液、1.5ミリメートルのMg 2とTaq DNAポリメラーゼ（Qiagen）を用いて反応液Clを、94℃で変性を35サイクルと0.2μMのプライマー℃で10秒間、40℃15秒のアニーリング℃で細菌の23Sリコン、および50℃、他のアンプリコン、72で拡張するための1分（72℃最終伸長℃で5分間）℃で。 </li><li> 1％アガロースゲルでPCR産物を表示します。 </li><li> 50μlの溶出バッファーで溶出をQIAクイックPCR精製キットを用いてPCR産物を精製する。光度計を用いて濃度を定量化する。 </li></ol></li><li>ビオチン標識アンチセンスのrRNAプローブ（arRNA） の in vitro転写。注：このステップでは、様々なrRNAのアンプリコンからサンプル特異的なプローブをMEGAscript T7キットを用いて合成される。 <ol><li>成分（ 表2）の下に混合することにより、各サンプル固有のrRNAのアンプリコンのための20μlの反応を設定します。 37℃で一晩インキュベート℃、 <stroNG&gt;注：通常、反応収率&gt; 50μgのRNA。 </li><li> DNAテンプレートを削除するには20分間37℃でインキュベートし、反応を1μlのDNase I（キットに含まれる）を追加します。 </li><li> ℃をarRNAを合成した沈殿物に4℃で10分間12,000 xgで遠心分離し、反応液に100μlの100％エタノールを加える。上清を捨て、750μlの75％エタノールでペレットを洗浄する。 50μlのヌクレアーゼフリー水でRNAプローブを再懸濁します。 </li><li>光度計を用いてRNA濃度を定量化する。 -80ストアプローブ℃、 </li></ol></li><li>ビオチン化arRNA rRNAとを減算。注：このステップは、全RNAサンプルでrRNAにハイブリダイズするビオチン化arRNAを採用しています。 rRNAをarRNAハイブリッドをストレプトアビジンコート磁気ビーズによってキャプチャされます。手順はマイナーな修正と参考文献13に記載されたプロトコルに基づいています。 <ol><li>試薬<ol><li> 0.1NのNaOH、20Xナトリウム塩素を準備IDE-クエン酸ナトリウム（SSC）と20％ホルムアミドで1×SSCで。 </li></ol></li><li>交配<ol><li>細菌の16Sおよび23Sプローブ（0.75μgの各々）、蚊の18Sおよび28Sプローブ（0.75μgの各々）と1μgの全RNA、1μlのRNase阻害剤、2.5μlの20X SSC緩衝液、200μlのPCRチューブに10μlの100％ホルムアミドを混ぜる、50μlにヌクレアーゼフリー水を追加します。 </li><li> 70℃のサーマルサイクラーとインキュベートに反応管を置き℃、5分間は、二次構造を開き、℃で1分間℃刻み各°Cの5を使用してrRNAおよびarRNAプローブのハイブリダイゼーションを可能にするために25に下降する。 </li></ol></li><li> rRNAの除去<ol><li> 1.7ミリリットルチューブに300μlのストレプトアビジンコーティングビーズを転送します。ビーズを固定化するために磁気分離ラックにチューブを置きます。上清を除去します。 0.1N NaOHおよびマイル300μlのビーズを再懸濁よくX。磁気ラックに戻ってチューブを配置し、上清をピペットで。 1X SSC300μlのビーズを再懸濁し、ビーズを固定し、上清をピペットで、よく混ぜる。 1X SSCの1以上の時間300μlの洗浄を繰り返します。 </li><li> 1.7ミリリットルチューブに、ビーズの3アリコート、それぞれ100μlを加える。磁気ラックにチューブを入れ、ピペッティングにより上清を除去する。注：ビーズのアリコートをビオチン化したrRNAをarRNAハイブリッドを捕捉する3ラウンドのために使用されます。 </li><li> 50μlのハイブリダイゼーション反応に20％ホルムアミド、50μlの1×SSCを追加します。第1ビードアリコートチューブに反応を移し、よく混ぜます。ビオチン化プローブ-rRNAのハイブリッドがストレプトアビジンビーズによって捕捉されるように室温で10分間インキュベートします。インキュベーション中に混在させるチューブを2分毎にフリックします。 </li><li>ビーズを固定化するために磁気ラックにチューブを置き、第二aliqに上清を移すビーズのUOTは、よく混ぜて、上記のようにインキュベートする。ビーズの第三チューブに上清を移すことによって第三のキャプ​​チャを行っています。混和後、。 </li><li>製造元のマニュアルに従ってホルムアミドを除去するためのRNeasy MinEluteキットを使用して、新しいチューブと行動の浄化に非rRNAの上清を移す。 </li><li>バイオアナライザ（ 図4）のAgilent RNA 6000ピコチップキットを用いることによりrRNAの枯渇の効率を確認してください。 </li></ol></li></ol></li></ol> 注：rRNAの枯渇したRNAサンプルが8必要に応じて増幅をmRNAに加算されます。

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著者らは、彼らが競合する経済的利益を持っていないことを宣言します。

複雑な微生物群集は、蚊の腸内生態系14,16,17に格納されています。 Metatranscriptomicシング（RNA-seq）は全体の微生物のトランスクリプトーム4,18を問い合わせることによってコンテキスト依存機能情報を明らかにすることができます。技術的には、原核生物のmRNAのオリゴd（T）媒介濃縮が原因でメッセンジャーの安定したポリ（A）尾の不在には適用されません。別の方法として、...

次世代シーケンシング技術は大きく分類学的組成や微生物集団の遺伝的機能を評価できるようにすることで、メタゲノム研究を進めてきた。 RNA-シーケンシング（RNA-seq）が1は、異なる文脈2-5に微生物metatranscriptomesを調査するために、培養ベースのメソッドをバイパスすることができます。原核生物のmRNA種を安定的ポリアデニル化されていないため、微生物のRNA-seqの大きな障害とは、mRNAを濃縮することが困難であることである。したがって、オリゴd（T）媒介メッセンジャー濃縮は適用されません。豊富なrRNAの除去は、mRNAを豊かにするための代替的なアプローチです。そのようなMicroexpress細菌のmRNA濃縮キット（Ambion）、RiboMinusトランアイソレーションキット（細菌）（Life Technologies）を、優先的エキソヌクレアーゼでrRNAを劣化させるのmRNA-ONLY原核mRNAアイソレーションキット（エピセンター）などの商業rRNAの枯渇キットは、使用されてきたrRNAの6-8を取り除くため。 Microexpressでしかし、捕捉プローブまたはRiboMinusは未知の微生物から典型的なグラム陽性菌およびグラム陰性菌から知られているrRNAを除去するための良い（メーカーの仕様を参照してください）​​が、リボソームRNAとの互換性に欠けています。その結果、除去はメタゲノムサンプル8-10にはあまり効率的であるかもしれません。エキソヌクレアーゼ処理が11を塗布した場合に加えて、mRNA存在量の忠実度には疑問だった。全体的に、減算ベースrRNAの枯渇がより少ないバイアスとメタゲノムの設定10-13のmRNAの濃縮度のより効果的であった。 蚊の腸はダイナミック微生物群集14が収容されている 。我々は、RNA-Seqを使って蚊腸microbiomeの機能を特徴付けることに興味を持っています。蚊の根性から単離されたRNAサンプルでは、​​蚊や微生物RNAの両方が存在している。ここでは、効率的にサブトラクティブハイブリダイゼーションにより、蚊や微生物rRNAを除去するために、地域社会固有のrRNAのプローブを使用するように修正されたプロトコルを記述します。得られたmRNA濃縮されたサンプルは、RNA - seqのに適しています。ワークフロー全体を図1に描かれている。

<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

蚊の腸は昆虫のライフサイクルのさまざまな段階でダイナミック微生物群集を収容する。腸コミュニティの遺伝能力と機能の特徴づけは、蚊生活特性に及ぼす腸内細菌叢に及ぼす影響への洞察を提供します。メタゲノムRNA-Seqは微生物群集中に存在する様々な微生物からトランスクリプトームを解析するための重要なツールとなっている。 rRNAは約90％を占めながら、メッセンジャーRNAは通常、全RNAのわずか百分の1から3を備えている。それは、ほとんどの原核生物のmRNA種が安定してポリ（A）尾部を欠いているため、メタゲノム微生物RNAサンプルからメッセンジャーRNAを豊かにするために挑戦している。これは、オリゴd（T）媒介mRNA分離を防ぐことができます。ここでは、メタゲノムトータルRNAサンプルからrRNAを除去するためにrRNAの捕獲プローブ由来の試料を用いてプロトコルを記述します。開始するには、両方の蚊と微生物、大小のサブユニットrRNAの断片は、メタゲノムコミュニティのDNAサンプルから増幅されています。その後、コミュnity特異的なビオチン化アンチセンスリボソームRNAプローブは、T7 RNAポリメラーゼを用いてin vitro で合成される。ビオチン化されたrRNAのプローブは、全RNAにハイブリダイズさせる。ハイブリッドは、ストレプトアビジンコートビーズによって捕獲され、全RNAから削除されます。この減算ベースのプロトコルでは、効率的にtotal RNAサンプルから蚊や微生物rRNAの両方を削除します。 mRNAを濃縮試料をさらにRNA増幅およびRNA - seqのために処理される。

（2）サンプル特異的捕捉のrRNAプローブの作成、rRNAの（3）枯渇サブトラクティブハイブリダイゼーションによる総RNAからrRNAをPCR用のメタゲノムDNA鋳型の製造（1）：このプロトコルは、次の3つのセクションが含まれています。高品質メタゲノムDNAとRNAの単離は、全体のプロセスに不可欠です。修正されたメタG-ノームDNA単離プロトコルは、 図2に示すように、蚊根性から高品質?...

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

リボソームRNA（rRNA）の枯渇プロトコルは蚊腸metatranscriptomeのRNA-seqのためのメッセンジャーRNA（mRNA）を豊かにするために開発されました。引き算を介しrRNAを除去するために使用されたサンプル特定のrRNAプローブは、蚊とその腸内微生物から作成されています。プロトコルのパフォーマンスは、rRNAの約90から99パーセントの除去をもたらすことができます。

モスキートガットメタゲノムRNA-seqのためのリボソームRNAの枯渇

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 ビュー.  New Mexico State University. リボソームRNA（rRNA）の枯渇プロトコルは蚊腸metatranscriptomeのRNA-seqのためのメッセンジャーRNA（mRNA）を豊かにするために開発されました。引き算を介しrRNAを除去するために使用されたサンプル特定のrRNAプローブは、蚊とその腸内微生物から作成されています。プロトコルのパフォーマンスは、rRNAの約90から99パーセントの除去をもたらすことができます?...

ビデオ: Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

シロアリの腸内微生物（Zootermopsis Angusticollis）からDNAを抽出し、腸内微生物の可視化

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

生物学

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細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

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システムの酵素のための基質の生成

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のトロンビン処理におけるトランスクリプトームの解析と制御ヒト肺微小血管内皮細胞

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リボソームRNA遺伝子アンプリコンの次世代シーケンシング

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.

アジアのタイガーモスキートにおける日長休眠のRNA-seqの分析のための実験とバイオインフォマティクス·プロトコル、<em&gt;ヒトスジシマカ</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.

生理的録音と黄熱病の蚊の味覚付属器RNAの配列決定<em&gt;ネッタイシマカ</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.

病気のベクトル蚊幼虫におけるキトサン/干渉RNAナノ粒子媒介遺伝子サイレンシング

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

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳類細胞における地上州枯渇超解像イメージング

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: 細胞型特異エピゲノムのプロファイリング

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...