이 작품은 NIH 보조금 1SC2GM092789-01A1에서 지원하고, MS는 NMSU 하워드 휴즈 의학 연구소 학부 연구 프로그램의 연구 학자였습니다. 동영상이 감독 제작 에이미 Lanasa 의해 NMSU의 크리에이티브 미디어 연구소와 박사 필립 루이스에 의해 조정되었다.

순서 1. 모기 양육 <ol><li> 27.5에서 insectary의 후면 모기 아노펠레스 gambiae G3 변형 ° C 어두운 / 빛의 80 % 습도와 12시 12분 시간주기에. </li><li> 비율 1시 1분에서 맥주의 효모와 지상 고양이 먹이와 애벌레 먹이를. </li><li> 3 일 계란 생산을위한 사후 출현에서 마우스의 피를 성인 모기를 먹이. </li></ol> 2. 모기 내장 해부 <ol><li> 압력솥 분해 도구 (슬라이드와 집게). </li><li> 흡인기를 사용하여 50 모기를 수집하고 CO 2 흐름 침대 (궁극적 Flypad)에 올려 놓습니다. 모기 몸 표면을 청소 70 % 에탄올을 포함하는 세 배양 접시에 순차적으로 모기 표본을 씻어. </li><li> stereomicroscopy 아래에 유리 슬라이드 표본을 놓으십시오. 내장을 제거합니다. </li><li> metagenomic DNA 절연을위한 조건 당 50 배짱를 수집합니다. metagenomic RNA 격리에 대한 조건 당 50 배짱를 수집합니다. </li></ol&gt; 3. Metagenomic DNA 절연 참고 : Metagenomic의 DNA는 아래에 설명 된 수정과 메타 - G-노움 DNA 절연 키트 (Epicentre의 Biotechnologies)를 사용하여 절연되어 있습니다. <ol><li> 300 μl TE에서 50 모기 용기를 놓습니다. 얼음에 속도 2,000 rpm으로 바이오 세대 PRO200 균질 (PRO 과학 Inc의, USA)를 사용하여 1 분 그들에게 균질화. </li><li> 바로 Lyse 라이소자임 솔루션 2 μl와 RNase 세포 현탁액에 1 μl를 추가합니다. 30 분에 37 ° C에서 vortexing하고 배양하여 섞는다. </li><li> 메타 용해 용액 (2X)와 튜브에 Proteinase K 1 μl 300 μl를 추가합니다. vortexing으로 섞는다. 간단히 솔루션의 모든 튜브의 바닥에있는 것을 보장하기 위해 튜브를 펄스 원심 분리기, 3 ~ 5 분을위한 얼음 후 실온에 시원한 15 분, 장소를 65 ° C에서 알을 품다. </li><li> 10 초에 힘차게 vortexing하여 관과 믹스에 MPC의 단백질 침전 시약의 350 μl를 추가합니다. </li><li> 펠릿 t4에 그는 12,000 XG에서 10 분을위한 원심 분리에 의한 파편 ° C. </li><li> 깨끗한 2 ML 튜브에 표면에 뜨는을 전송하고, 펠릿 폐기합니다. </li><li> 표면에 뜨는에 이소프로판올의 570 μl를 추가합니다. 반전 튜브를 여러 번에 섞는다. </li><li> 펠렛 4에 12,000 XG에서 10 분을위한 원심 분리하여 DNA ° C. DNA 펠렛을 dislodging없이 이소프로판올을 제거합니다. </li><li> 펠렛을 씻으 75 % 에탄올 500 μl를 추가합니다. 4 ° C.에서 12,000 XG에서 5 분 동안 원심 분리기 DNA 펠렛을 방해하지 않으면 서 에탄올을 제거합니다. 2 분 동안 실온에서 펠렛을 공기 건조. 참고 : DNA 펠렛 이상 건조하지 마십시오. </li><li> TE 버퍼 50 μl의 DNA 펠렛을 Resuspend. </li><li> 1 % 아가로 오스 겔의 겔 전기 영동을 통해 키트에서 제공하며 Fosmid 제어 DNA (100 NG / μl 40킬로바이트)에 비교하여 절연 DNA의 크기를 확인합니다. </li></ol> 4. 총 RNA 절연 참고 : 작업 영역을 청소RNase 오염을 최소화하기 RNaseZap을 갖추고 있습니다. Metagenomic RNA는 사소한 수정으로 TriPure 절연 시약 (로슈)를 사용하여 모기 내장 표본으로부터 격리되었다. <ol><li> 500 μl TriPure 절연 시약과 2 ML 튜브에 50 모기 용기를 놔. 균질를 사용하여 얼음에 속도 2,000 rpm에서 1 분 동안 균질화. 핵 단백질 단지의 분리를 허용하도록 5 분에 RT에 앉아 보자. </li><li> 12 초 100 μl 클로로포름과 소용돌이를 추가하고 2 분에 RT에 앉아 보자. </li><li> 10 분 10,000 XG에 원심 분리기. 조심스럽게 불쾌감을 분리 단계없이 튜브를 꺼내. </li><li> 새로운 튜브에 수성 단계를 전송, 250 μl 이소프로판올을 추가 잘 혼합하고, 20 분에 -20 ° C에 넣어. </li><li> 4에 10 분에 12,000 XG에 원심 분리기 ° C 석출물 RNA합니다. </li><li> 펠렛을 dislodging하지 않고 표면에 뜨는 폐기하십시오. 750 μl 75 % 에탄올로 펠렛을 씻으십시오. </li><li> 펠렛을 방해하지 않고 표면에 뜨는을 피펫.IR은 2 분의 튜브를 건조. </li><li> 30 μl nuclease 무료 물로 RNA를 Resuspend. </li><li> 20 분에 37 ° C에서 DNase와 총 RNA를 처리 한 후 75 잠복기에 의해 DNase를 비활성화 15 분에 ° C. 30 μl nuclease 무료 물에 에탄올과 resuspend RNA와 석출물 RNA. </li><li> NanoDrop를 사용하여 총 RNA의 양을 결정합니다. </li></ol> 5. Ribosomal RNA 고갈 참고 :이 프로토콜은 이전에 설명한 방법 12,13,15에 따라 개발되었습니다. <ol><li> ribosomal RNA 유전자 조각의 PCR 증폭 이 단계는 전체 RNA 샘플에서 rRNA 할 무료입니다 안티 - 감각 ribosomal RNA (arRNA) 프로브를 생산하는 체외 스크립트에 대한 템플릿으로 사용됩니다 샘플 별 rRNA 풀을 만듭니다. <ol><li> rRNA 조각 PCR (표 1)에 대한 프라이머 세트를 디자인하고 합성. </li><li> (50)에서 PCR을 실행50 NG DNA 템플릿, 1 개 PCR 버퍼, 1.5mM MG 2 DNA 형성 촉매의 DNA 중합 효소 (Qiagen)를 사용하여 μl 반응 망할 CIA, 94에서 denaturing의 35주기와 0.2 μM 프리 머 ° C 10 초에 40에서 15 초 어닐링 ° C에 박테리아 23S amplicon, 50 ° C, 다른 amplicons, 72의 확장을위한 1 분 (72시 최종 확장 ° C 5 분)을위한 ° C. </li><li> 1퍼센트 아가로 오스 겔에 PCR 제품을 볼 수 있습니다. </li><li> 50 μl 용출 버퍼에 용출과 QIAquick PCR 정화 키트를 사용하여 PCR 제품을 정화. NanoDrop를 사용하여 농도를 정량화. </li></ol></li><li> 비오틴 - 표시 안티 센스 rRNA 프로브 (arRNA)의 체외 스크립트합니다. 참고 :이 단계에서, 다양한 rRNA의 amplicons에서 샘플 특정 프로브는 MEGAscript T7 키트를 사용하여 합성합니다. <ol><li> 아래의 재료 (표 2) 혼합하여 각 샘플 특정 rRNA의 amplicon을위한 20 μl 반응을 설정합니다. 37 박 품다 ° C. <stroNG&gt; 참고 : 일반적으로 반응 수율&gt; 50 μg의 RNA. </li><li> DNA 템플릿을 제거 할 20 분에 37 ° C에서 반응과 배양에 1 μl DNase I을 (키트에 포함)를 추가합니다. </li><li> 4에 10 분에 12,000 XG에서 반응 100 μl 100 % 에탄올, 원심 분리기를 추가 ° C arRNA을 합성 침전 할 수 있습니다. 표면에 뜨는 무시하고 750 μl 75 % 에탄올로 펠릿을 씻는다. 50 μl nuclease 무료 물에 Resuspend RNA 프로브. </li><li> NanoDrop를 사용하여 RNA 농도를 정량화. -80에서 저장 프로브 ° C. </li></ol></li><li> biotinylated arRNA과 rRNA 빼기. 참고 :이 단계는 전체 RNA 샘플에서 rRNA에 잡종을 만들다 할 수있는 biotinylated arRNA을 사용합니다. rRNA-arRNA의 하이브리드는 streptavidin - 코팅 자성 구슬에 의해 캡처됩니다. 절차는 약간의 수정을 참조 13에 설명 된 프로토콜을 기반으로합니다. <ol><li> 시약 <ol><li> 0.1 N NaOH, 20X 나트륨 chlor 준비IDE - 시트르산 (SSC)와 20 %의 포름 아미드 1 X SSC. </li></ol></li><li> 이종 교잡 <ol><li> 세균 16와 23S 프로브 (0.75 μg 각), 모기 18S와 28S 프로브 (0.75 μg 각) 1 개 μg 총 RNA를 섞어, 1 μl RNase 억제제, 2.5 μl 20X SSC 버퍼 200 μl PCR 튜브에 10 μl 100 % 포름 아미드 50 μl에 추가 nuclease - 무료 물. </li><li> 70에서 열 자전거 타는 사람 및 배양에 반응 튜브를 놓습니다 ° C 5 분 거리에 보조 구조를 열고 25 다운 방면 진입로에 대한 ° C rRNA와 arRNA 프로브의 하이브리드를 허용하도록 1 분마다 °의 C에 5 ° C 단위를 사용합니다. </li></ol></li><li> rRNA의 제거 <ol><li> 1.7 ML 튜브에 300 μl streptavidin 코팅 구슬을 전송합니다. 구슬을 고정 할 수있는 자기 분리 랙에 튜브를 놓습니다. 표면에 뜨는 제거합니다. 0.1N NaOH와 미 300 μl의 구슬을 Resuspend잘 X. 자기 랙에 다시 튜브를 삽입하고 표면에 뜨는을 피펫. 잘 혼합, 1X SSC 300 μl의 구슬을 Resuspend, 구슬을 고정하고 표면에 뜨는을 피펫. 1X SSC 한 번 더 300 μl로 세탁을 반복합니다. </li><li> 구슬 3 aliquots, 100 μl 각각 1.7 ML 튜브의하십시오. 자기 랙에 튜브를 삽입하고, pipetting하여 표면에 뜨는을 제거합니다. 참고 : 구슬의 aliquots는 biotinylated rRNA-arRNA의 하이브리드를 캡처하는 3 라운드에 사용됩니다. </li><li> 50 μl 하이브 리다이 제이션 반응에 20 % 포름 아미드와 50 μl 1X SSC를 추가합니다. 첫 번째 구슬 나누어지는 관에 반응을 전송하고, 잘 섞는다. biotinylated 프로브-rRNA의 하이브리드는 streptavidin 구슬에 의해 캡처 할 수 있도록하기 위해 10 분 동안 RT에서 알을 품다. 관에게 부화하는 동안 혼합 할 때마다 2 분 영화. </li><li> 구슬을 고정하기 위해 자기 랙에 튜브를 놓고, 두 번째 aliq로 표면에 뜨는 전송비즈 uot는 잘 혼합 위 품다. 구슬의 세 번째 관으로 표면에 뜨는을 전송하여 세 번째 캡처를 실시하고 있습니다. 믹스 앤 품다. </li><li> 제조업체의 설명서에 따라 포름 아미드를 제거하는 RNeasy MinElute 키트를 사용하여 새 튜브 및 행동 정화에 비 rRNA 표면에 뜨는을 전송합니다. </li><li> Bioanalyzer (그림 4)에 애질런트 RNA 6000 피코 칩 키트를 사용하여 rRNA 고갈 효율을 확인합니다. </li></ol></li></ol></li></ol> 참고 : rRNA 소모 RNA 샘플은 8 필요한 경우 증폭을 mRNA 될 수 있습니다.

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservative+fragments+in+bacterial+16S+rRNA+genes+and+primer+design+for+16S+ribosomal+DNA+amplicons+in+metagenomic+studies.">Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.</a> PLoS One. 4, e7401 (2009).</li><li>Hunt, D. E., 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=Evaluation+of+23S+rRNA+PCR+primers+for+use+in+phylogenetic+studies+of+bacterial+diversity.">Evaluation of 23S rRNA PCR primers for use in phylogenetic studies of bacterial diversity.</a> Appl. Environ. Microbiol. 72, 2221-2225 (2006).</li></ol>

제작자들은 더 경쟁 금융 이익이 없다는 점 선언합니다.

복잡한 미생물 커뮤니티는 모기 내장 생태계 14,16,17에 있습니다. Metatranscriptomic 시퀀싱 (RNA-seq)는 전체 미생물의 transcriptome 4,18을 조사하여 상황에 따라 기능 정보를 공개 할 수 있습니다. 기술적으로는, prokaryotic mRNA의 oligo-D (T) 매개 농축 인해 안정적인 폴리 메신저의 (A) 꼬리의 부재에 적용되지 않습니다. 또한, rRNA의 고갈은 mRNA 농축에 사용되었습니다. 여기, 우리는 모기 내장 미?...

차세대 시퀀싱 기술은 크게 분류 학적 조성과 미생물 조립의 유전자 기능을 평가 할 수 있도록하여 metagenomics 연구를 전진했다. RNA-시퀀싱 (RNA-Seq) 1 서로 다른 상황 2-5에서 미생물 metatranscriptomes를 조사 문화 기반 방법을 무시할 수 있습니다. prokaryotic mRNA 종은 안정적으로 polyadenylated되지 않으므로 미생물 RNA-seq에 대한 주요 장애물은 mRNA을 풍요롭게에 어려움이 있습니다. 따라서, oligo D (T) 매개 메신저 농축이 적용되지 않습니다. 풍부한 rRNA의 제거는 mRNA을 풍부하게 할 수있는 대안 방법입니다. 이러한 Microexpress 박테리아 mRNA 농축 키트 (Ambion), RiboMinus Transcriptome 절연 키트 (박테리아) (생명 기술), 그리고 우선적으로 exonuclease와 rRNA을 방해 mRNA-ONLY Prokaryotic mRNA 절연 키트 (Epicentre)와 같은 상업 rRNA 고갈 키트는 사용되었다 rRNA 6-8를 제거하십시오. Microexpress에서 그러나, 캡처 프로브또는 RiboMinus은 (제조업체의 사양을 참조) 전형적인 그람 양성과 그람 음성 세균에서 알려진 rRNA를 제거에 좋은,하지만 알 수없는 미생물의 rRNA와 덜 호환됩니다. 따라서, 제거 metagenomic 샘플 8-10에 덜 효율적으로 할 수 있습니다. exonuclease 치료가 11을 적용했을 때 또, mRNA의 풍부한의 충실도 의심했다. 전반적으로, 뺄셈 기반 rRNA 고갈 적은 편견과 metagenomic 설정 10-13의 mRNA 농축에 더 효과적이었습니다. 모기 창자는 역동적 미생물 커뮤니티 14 숙박이 가능합니다. 우리는 RNA-Seq을 사용하여 모기 내장 microbiome의 기능을 특성화에 관심이 있습니다. 모기 내장 격리 RNA 샘플에서 모두 모기와 미생물의 RNA가 존재한다. 여기, 우리는 효율적으로 subtractive 하이브리드에 의해 모기와 미생물 rRNA를 고갈 지역 사회 특정 rRNA 프로브를 사용하는 수정 된 프로토콜을 설명합니다. 결과 mRNA농축 샘플 RNA-Seq에 적합합니다. 전체 흐름은 그림 1에 묘사되어 있습니다.

<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

모기 직감은 곤충의 생활주기의 다른 단계에 걸쳐 동적 미생물 커뮤니티를 수용 할 수 있습니다. 내장 커뮤니티의 유전 용량과 기능의 특성은 모기 삶의 특성에 내장 microbiota의 효과에 대한 통찰력을 제공 할 것입니다. Metagenomic RNA - Seq는 미생물 지역 사회에 존재하는 다양한 미생물에서 transcriptomes을 분석 할 수있는 중요한 도구가되었습니다. rRNA는 약 90 %를 구성하는 동안 메신저 RNA는 일반적으로, 총 RNA의 1-3 위에 만 %를 포함한다. 대부분의 prokaryotic mRNA 종은 안정 폴리 (A) 꼬리 부족 때문에 metagenomic 미생물 RNA 샘플에서 메신저 RNA를 풍부하게 도전하고 있습니다. 이 oligo D (T) 매개의 mRNA 절연을 방지합니다. 여기, 우리는 rRNA가 metagenomic 총 RNA 샘플에서 rRNA를 제거하는 프로브를 캡처 파생 샘플을 사용 프로토콜을 설명합니다. 시작하려면, 모기와 미생물의 크고 작은 subunit의 rRNA 조각 모두 metagenomic 지역 사회의 DNA 샘플에서 증폭됩니다. 그런 다음, community 특정 biotinylated 안티센스 RNA ribosomal 프로브는 T7 RNA 중합 효소의를 사용하여 체외에서 합성되어 있습니다. biotinylated rRNA 프로브는 총 RNA에 hybridized 있습니다. 하이브리드는 streptavidin - 코팅 구슬에 의해 캡처 된 총 RNA에서 제거됩니다. 이 빼기 기반 프로토콜을 효율적으로 전체 RNA 샘플에서 모기와 미생물 rRNA를 모두 제거합니다. mRNA 농축 샘플은 또한 RNA의 증폭 및 RNA-Seq에 처리됩니다.

(2) 샘플 특정 캡처 rRNA 프로브를 생성, rRNA (3) 고갈 subtractive 하이브리드의 총 RNA의 rRNA PCR을위한 metagenomic DNA 템플릿 (1) 준비 : 프로토콜은 세 부분이 포함되어 있습니다. 고품질 metagenomic DNA와 RNA의 분리는 전체 프로세스에 필수적입니다. 수정 메타 G-노움 DNA 격리 프로토콜은 그림 2에 표시된 모기 내장에서 높은 품질의 metagenomic DNA를 산출. 그것은 모기 내장에서 높은 품질의 총 RNA를 ?...

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

ribosomal RNA (rRNA) 고갈 프로토콜은 모기 내장 metatranscriptome의 RNA-seq를위한 메신저 RNA를 (mRNA) 풍부하게하기 위해 개발되었습니다. 뺄셈을 통해 rRNA를 제거하는 데 사용 된 샘플 특정 rRNA 프로브은, 모기와 내장 미생물에서 만들어졌습니다. 프로토콜의 성능은 rRNA의 약 90~99%의 삭제 될 수 있습니다.

모기 내장 Metagenomic RNA-seq에 대한 Ribosomal RNA의 고갈

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 조회수.  New Mexico State University. ribosomal RNA (rRNA) 고갈 프로토콜은 모기 내장 metatranscriptome의 RNA-seq를위한 메신저 RNA를 (mRNA) 풍부하게하기 위해 개발되었습니다. 뺄셈을 통해 rRNA를 제거하는 데 사용 된 샘플 특정 rRNA 프로브은, 모기와 내장 미생물에서 만들어졌습니다. 프로토콜의 성능은 rRNA의 약 90~99%의 삭제 될 수 있습니다.

비디오: 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.

마이트의 굿 미생물 (Zootermopsis Angusticollis)에서 DNA를 추출하고 굿 미생물을 떠올리

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 ...

연구

생물학

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 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

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 / 카스 시스템의 Endonucleases을위한 기판 생성

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-seq의 트롬빈 - 처리의 Transcriptomes 분석 및 제어 인간 폐 Microvascular 내피 세포

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 리보솜 RNA 유전자 증폭 산물의 차세대 시퀀싱

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.

아시아 타이거 모기에 Photoperiodic 휴면의 RNA-서열 분석을위한 실험 및 생물 정보학 프로토콜,<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.

생리 녹음 및 황열병 모기의 미각 부속 기관의 RNA 시퀀싱<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.

질병 벡터 모기 유충의 키토산 / 간섭 RNA 나노 입자 중재 유전자 음소거

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.

포유류 세포에 있는 바닥 상태 고갈 슈퍼 해상도 영상

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: 셀 형식 관련 Epigenome 프로 파일링

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