Name: a method for measuring rna n6-methyladenosine modifications in cells and tissues
Uploaded: 2016-12-05T13:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

C.Y.W. received support from the National Health Research Institute (NHRI-EX101-9925SC), the National Science Council (101-2314-B-182-100-MY3, 101-2314-B-182A-009), and Chang Gung Memorial Hospital (CMRPG3B1643, CMRPG3D1002, CMRPG3D0581, CMRPG380091, and CMRPG3C1763).

참고 : 6 수준은 rRNA의, mRNA를, 및 기타 소형의 RNA를 포함 총 RNA의 m. 리보솜 RNA 풍부한 m 6 수정,도 6a 레벨이 사실을 고려할 필요가 m의 측정을 가지고 있기 때문이다. 1. RNA 분리 <ol><li> (종이 타월에 분사)가 리보 뉴 클레아 제의 오염 제거 용액을 사용하여, 피펫 및 실험실 벤치의 표면을 닦으십시오. 젖은 종이 클레아없는 물과 수건을 다시 피펫 및 실험실 벤치 표면을 닦아. </li><li> RNA 추출 <ol><li> 총 RNA 분리 <ol><li> 오버 헤드 교반기를 사용하여, 조직을 50 내지 100 mg을 4 ℃에서 유지 RNA 분리 액 1ml에 5 내지 10 × 106 세포를 균질화시킨다. 주 : 자세한 조직 (100-150 mg)을하기 때문에 내부 하부 RNA 농도 생쥐 지방 조직 샘플에 대해 요구 될 수있다. 샘플은 마우스 심장, 간, 골격근, 폐, 뇌에서 공급하고, 대 식세포는 이전에 4 사용되어왔다. </li><li> 에서실온에서 5 분 동안 샘플을 균질화 cubate. </li><li> 샘플 파쇄 1 ㎖ 당 1- 브로 모 -3- 클로로 프로판 (BCP) 100 μL를 추가한다. 15 초 동안 적극적으로 튜브를 흔들 제조업체의 지침 (24)에 따라 총 RNA를 분리로 진행합니다. </li></ol></li><li> 격리 된 총 RNA에서 폴리아 데 닐화 mRNA의 정제 <ol><li> 사용 가능한 키트 또는 프로토콜을 사용하여 폴리아 데 닐화의 mRNA를 정화. <ol><li> 다음 솔루션의 각을 다시 중단 철저하게 흔들어 : 배 결합 솔루션, 올리고 (DT) 폴리스티렌 구슬 및 세척 솔루션을. 올리고 (DT)를 사용하기 전에 실온에 따뜻한 비즈 있는지 확인합니다. </li><li> 가열 블럭에서 70 ℃로 튜브 열로 제조 당 용출 용액 120 μL를 옮긴다. </li><li> 미세 원심 관에 총 RNA의 500 μg의 최대 피펫. 클레아없는 물로, 250 μL에 볼륨을 조정합니다. </li><li> 총 RNA에 2 배 결합 액 250 μL를 추가하기 때문에lution와 소용돌이 짧게 내용을 혼합합니다. </li><li> 올리고 15 μL를 추가 (DT) 구슬과 소용돌이는 철저하게 혼합합니다. </li><li> 3 분 동안 70 ℃에서 혼합물을 인큐베이션. </li><li> 가열 블록의 샘플을 제거하고 10 분 동안 실온에서 서하자. </li><li> 2 분 동안 15,000 XG에 원심 분리기. </li><li> 조심스럽게 뒤 약 50 μL를 떠나, 뜨는을 제거합니다. </li><li> 피펫으로 펠렛을 다시 중단 세척 용액 500 μL를 추가합니다. </li><li> 스핀 필터 / 수집 튜브 세트로 현탁액을 피펫. </li><li> 2 분 동안 15,000 XG에 원심 분리기. 포집 관에서 열을 제거하고 흐름을 통해 폐기하십시오. 수집 튜브에 열을 돌려줍니다. </li><li> 스핀 필터 상에 세척 용액의 피펫 500 μL. </li><li> 15,000 X 원심 분리기 2 분 동안 g. </li><li> 신선한 수집 관에 스핀 필터를 전송합니다. </li><li> 용출 용액의 피펫 50 μL 가열70 ° C 스핀 필터의 중심 상. </li><li> 70 ℃에서 2-5 분 동안 품어. 1 분 동안 15,000 XG에 원심 분리기. </li><li> 스핀 필터의 중심에 70 ° C로 가열 용출 용액의 추가 50 μL 피펫. </li><li> 단계를 반복 1.2.2.1.18. </li></ol></li><li> 100 μg의 / ㎖ 글리코겐, 3 M 아세트산 나트륨 완충액 (pH 5.2), 및 무수 에탄올 2.5 부피 0.1 볼륨. -80 ° C에서 하룻밤 침전. </li><li> 4 ℃에서 25 분 동안 15,000 XG에 원심 분리기. 상층 액을 버린다. </li><li> 75 % 에탄올 1 mL로 펠렛을 씻으십시오. 4 ℃에서 15 분 동안 15,000 XG에 원심 분리기. 조심스럽게 에탄올을 제거합니다. </li><li> 3-5 분 동안 공기 건조. </li><li> 펠렛을 용해 뉴 클레아없는 물 10 μL를 추가합니다. </li><li> -70 ° C에서 보관하십시오. </li></ol></li></ol></li><li> RNA 자격 및 정량화 <ol><li> 분광 광도계를 사용하여, NotI에 의해 RNA의 농도를 결정260 nm 내지 280 nm에서의 흡광도 겨. 다음은 260 / A 280 비율은 1.8-2.2해야합니다. </li><li> 0.8 % 아가 로스 겔 전기 영동 (25)를 사용하여 RNA 샘플 품질을 확인합니다. 참고 :이 그대로 진핵 총 RNA는 강렬한 28S와 18S rRNA의 밴드를 표시해야합니다. 좋은 RNA 준비를위한 18S rRNA의 밴드 강도를 비교 28S의 비율은 1 ~ 2이다. </li></ol></li></ol> 2. 젤 전기 영동 및 전송 참고 : 버퍼 준비 프로토콜은 표 1에 제시되어있다. <ol><li> 포름 알데히드 젤 준비 (1 %) <ol><li> 디 에틸 피로 카보 (DEPC) 물과 모든 전기 장비를 씻어. </li><li> 완전히 전자 레인지 DEPC 물 215 ㎖의 2.5 g 아가 용융. </li><li> 10 배 3- (N- 모르 폴리 노) 프로판 술폰산 (MOPS)의 12.5 mL의 버퍼와 37 %의 포름 알데히드 22.5 mL를 넣고. </li><li> 1.5 mm 두께의 빗 전기 영동 장치에 그것을 따르십시오. </li><li> t 거품을 제거 또는 푸시깨끗한 빗과 젤의 가장자리에 밑단. </li><li> 겔은 실온에서 응고 할 수 있습니다. </li></ol></li><li> 샘플 준비 <ol><li> 샘플 버퍼의 총 RNA의 1-10 μg의 11.3 μL를 섞는다. </li><li> 샘플 버퍼의 11.3 μL와 RNA 마커 (1 μg의 / μL) 2 μg의 혼합. </li><li> 16 μL의 총 볼륨 2.2.1과 2.2.2에서 샘플 뉴 클레아없는 물을 추가합니다. </li><li> 5 분 동안 60 ℃에서 가열 한 후 얼음에 진정. </li><li> 얼음 / μL 에티 디움 브로마이드 0.1 μg의를 포함 추적 염료의 4 μL와 2.2.3에서 샘플 16 μL를 섞는다. 주의 : 흡입하면 에티 디움 브로마이드 가능성이 기형 독성이다. 에티 디움 브로마이드 물질 안전 보건 자료를 참조하십시오. </li></ol></li><li> 전기 영동 <ol><li> 10 배 MOPS의 200 ㎖가 1 배 MOPS 실행 버퍼를 만들기 위해 DDH 2 O의 1,800 mL의 버퍼에 추가합니다. </li><li> 젤의 우물을 씻어하기 위해 1 배 MOPS 실행 버퍼를 사용합니다. </li><li> 사전 실행 t그는 5 분 동안 20 V에서 젤. </li><li> 겔의 우물에 RNA 샘플을로드합니다. </li><li> 빛의 노출을 방지하기 위해 호일로 커버. (야간) 약 17 시간 동안 35 V에서 실행 </li><li> 검사 및 자외선 아래에서 젤을 촬영. </li></ol></li><li> 이전 <ol><li> 사용되지 않는 부분을 제거하는 젤을 잘라. </li><li> 10 배 SSC 버퍼 (20 배 SSC 버퍼의 250 mL의 DEPC 물 250 mL)을 500 ㎖를 준비합니다. </li><li> 50 rpm으로 진탕 20 분 동안 10 배 SSC 버퍼로 두 번 젤을 씻으십시오. </li><li> 반송 트레이 (도 1)로부터 전송 버퍼를 흡수 심지 종이의 역할을하기에 충분히 큰 1 필터 종이 낱장. </li><li> 젤과 같은 크기로 종이 필터 4 조각을 잘라. </li><li> 겔과 같은 크기로 양전하 나일론 막 중 1 매를 잘랐다. </li><li> 겔의 노치와 올바른 방향을 보장하기 위해 마커로 막을 표시합니다. </li><li> 15 분 동안 2 × SSC 완충액에서 막을 담근다. </li><li> 500 mL의 O를 붓고F 10 배 SSC 전송 트레이에 버퍼. </li><li> 유리판을 가로 지르는 큰 필터 종이 시트를 놓고 전송 버퍼 여과지 젖은. </li><li> 피펫있는 모든 거품을 굴립니다. </li><li> 전송 버퍼에 미리 차단 필터 종이의 2 개를 적시 단계 2.4.5에서 여과지의 중앙에 배치합니다. 모든 거품을 제거합니다. </li><li> 아래 필터 종이에 젤 상단 측을 놓습니다. </li><li> 겔의 상단에 막을 놓습니다. 겔과 멤브레인의 노치에 맞 춥니 다. </li><li> 멤브레인 위에 프리 컷 여과지의 2 개를 배치하고 전송 버퍼 여과지 젖은. </li><li> 층 사이의 거품을 제거합니다. </li><li> 수건은 겔 멤브레인을 통과 버퍼를 흡수되도록하여 겔 주위에 랩을 적용합니다. </li><li> 필터 종이 위에 종이 타월 스택. </li><li> 수건 위에 대형 유리 접시를 놓습니다. </li><li> 스택의 상단에 무게를 둡니다. </li><li>RNA가 막에 겔로부터 전송할 수 밤새 유지. </li></ol></li></ol> N 6 -methyladenosine 3. 검출 <ol><li> 막 가교 <ol><li> 배 SSC 버퍼에 젖은 멤브레인을 유지합니다. </li><li> UV 가교제에 10 배 SSC 버퍼에 몰입 필터 종이 2 장을 놓습니다. </li><li> RNA의 흡착면이 위를 향하도록, 여과지 위에 멤브레인을 놓습니다. </li><li> &quot;autocrosslink 모드&quot;(12 μJ)를 선택하고 조사를 시작하기 위해 &quot;시작&quot;버튼을 누릅니다. </li><li> 멤브레인을 겔에서 RNA의 전사를 확인하기 위해 UV 겔 화상 포수 막 겔을 조사한다. </li></ol></li><li> 면역 <ol><li> 실온에서 1 시간 동안 트윈 -20 (TBST) 완충액 TBS 버퍼에서 5 % 무 지방 우유 막을 차단. </li><li> 15 분 동안 TBST 완충액으로 세 번 씻으십시오. </li><li> 밤새 m (6) A (에 멤브레인을 품어 &lt;EM&gt; N 6 -methyladenosine) 항체 용액 (1 : 1,000 5 % 무 지방 우유 TBST 버퍼) 4 ° C에서. </li><li> 15 분 동안 TBST 완충액으로 세 번 멤브레인을 씻으십시오. </li><li> 실온에서 1 시간 동안 (5 % 무 지방 우유 TBST 버퍼 2000 1) HRP 접합 된 당나귀 항 - 토끼 항체 용액의 막을 인큐베이션. </li><li> 15 분 동안 TBST 완충액으로 세 번 멤브레인을 씻으십시오. </li><li> 강화 된 화학 발광 기판 (막 cm 2 당 0.125 ml)에 적용합니다. </li><li> 제조자의 지시 (26)에있어서, 상기 디지털 영상 기의 최적 설정으로 화학 발광 캡처. </li></ol></li><li> m 6 정량화 <ol><li> ImageJ에 소프트웨어를 사용하여 상대 m 6 화학 발광 강도를 측정한다. <ol><li> ImageJ에 소프트웨어 메뉴 아래에서 해당 파일을 열 수있는 &quot;파일&quot;옵션을 선택합니다. </li><li> ImageJ에에서 &quot;직사각형&quot;도구를 선택하고 주변에 프레임을 그립니다신호. </li><li> 리보솜 RNA는 실험의 초점없는 경우 계산을위한 18S 및 28S 리보솜 RNA m도 6A 대역을 피한다. </li><li> 선택한 차선을 확인하는 &quot;명령&quot;과 &quot;1&quot;을 클릭합니다. </li><li> 보도는 &quot;명령&quot;과 &quot;3&quot;선택한 플롯을 표시합니다. </li><li> 가 &quot;STRAIGHT&quot;도구를 클릭하고 분할 영역을 구분하는 선을 그립니다. </li><li> 측정 값을 기록하기 위해 &quot;지팡이&quot;도구를 클릭합니다. </li><li> 데이터를 내 보냅니다. </li><li> 에티 디움 브로마이드 염색 중 18S 리보솜 RNA 밴드 m (6) (A)의 레벨을 정규화한다. </li></ol></li></ol></li></ol>

<ol>
	<li>Thammana, P., Held, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+16S+RNA+during+ribosome+assembly+in+vitro.">Methylation of 16S RNA during ribosome assembly in vitro.</a> Nature. 251 (5477), 682-686 (1974).</li><li>Meyer, K. D., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+epitranscriptome:+N6-methyladenosine+and+gene+expression+control.">The dynamic epitranscriptome: N6-methyladenosine and gene expression control.</a> Nat Rev Mol Cell Biol. 15 (5), 313-326 (2014).</li><li>Niu, Y., 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=N6-methyl-adenosine+(m6A)+in+RNA:+an+old+modification+with+a+novel+epigenetic+function.">N6-methyl-adenosine (m6A) in RNA: an old modification with a novel epigenetic function.</a> Genomics Proteomics Bioinformatics. 11 (1), 8-17 (2013).</li><li>Wang, C. Y., 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=Loss+of+FTO+in+adipose+tissue+decreases+Angptl4+translation+and+alters+triglyceride+metabolism.">Loss of FTO in adipose tissue decreases Angptl4 translation and alters triglyceride metabolism.</a> Sci Signal. 8 (407), 127 (2015).</li><li>Wang, X., 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=N6-methyladenosine-dependent+regulation+of+messenger+RNA+stability.">N6-methyladenosine-dependent regulation of messenger RNA stability.</a> Nature. 505 (7481), 117-120 (2014).</li><li>Berulava, T., Rahmann, S., Rademacher, K., Klein-Hitpass, L., Horsthemke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N6-adenosine+methylation+in+MiRNAs.">N6-adenosine methylation in MiRNAs.</a> PLoS One. 10 (2), 0118438 (2015).</li><li>Deng, X., 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=Widespread+occurrence+of+N6-methyladenosine+in+bacterial+mRNA.">Widespread occurrence of N6-methyladenosine in bacterial mRNA.</a> Nucleic Acids Res. 43 (13), 6557-6567 (2015).</li><li>Dominissini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+N-methyladenosine+methylome+in+eukaryotic+messenger+RNA.">The dynamic N-methyladenosine methylome in eukaryotic messenger RNA.</a> Nature. , (2016).</li><li>Fustin, J. M., 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=RNA-methylation-dependent+RNA+processing+controls+the+speed+of+the+circadian+clock.">RNA-methylation-dependent RNA processing controls the speed of the circadian clock.</a> Cell. 155 (4), 793-806 (2013).</li><li>Geula, S., 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=Stem+cells.+m6A+mRNA+methylation+facilitates+resolution+of+naive+pluripotency+toward+differentiation.">Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.</a> Science. 347 (6225), 1002-1006 (2015).</li><li>Wang, C. Y., 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=FTO+modulates+fibrogenic+responses+in+obstructive+nephropathy.">FTO modulates fibrogenic responses in obstructive nephropathy.</a> Sci Rep. , (2016).</li><li>Jia, G., 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=N6-methyladenosine+in+nuclear+RNA+is+a+major+substrate+of+the+obesity-associated+FTO.">N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.</a> Nat Chem Biol. 7 (12), 885-887 (2011).</li><li>Du, T., 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=An+association+study+of+the+m6A+genes+with+major+depressive+disorder+in+Chinese+Han+population.">An association study of the m6A genes with major depressive disorder in Chinese Han population.</a> J Affect Disord. 183, 279-286 (2015).</li><li>Wang, C. Y., Yeh, J. K., Shie, S. S., Hsieh, I. C., Wen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+RNA+N6-methyladenosine+and+the+role+of+cryptochrome.">Circadian rhythm of RNA N6-methyladenosine and the role of cryptochrome.</a> Biochem Biophys Res Commun. 465 (1), 88-94 (2015).</li><li>Wang, C. Y., Shie, S. S., Hsieh, I. C., Tsai, M. L., Wen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FTO+modulates+circadian+rhythms+and+inhibits+the+CLOCK-BMAL1-induced+transcription.">FTO modulates circadian rhythms and inhibits the CLOCK-BMAL1-induced transcription.</a> Biochem Biophys Res Commun. 464 (3), 826-832 (2015).</li><li>Heyn, H., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Adenine+Code+for+DNA:+A+Second+Life+for+N6-Methyladenine.">An Adenine Code for DNA: A Second Life for N6-Methyladenine.</a> Cell. 161 (4), 710-713 (2015).</li><li>Xiao, W., 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=Nuclear+m(6)A+Reader+YTHDC1+Regulates+mRNA+Splicing.">Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.</a> Mol Cell. 61 (4), 507-519 (2016).</li><li>Meyer, K. D., 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=Comprehensive+analysis+of+mRNA+methylation+reveals+enrichment+in+3'+UTRs+and+near+stop+codons.">Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.</a> Cell. 149 (7), 1635-1646 (2012).</li><li>Horowitz, S., Horowitz, A., Nilsen, T. W., Munns, T. W., Rottman, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+N6-methyladenosine+residues+in+bovine+prolactin+mRNA.">Mapping of N6-methyladenosine residues in bovine prolactin mRNA.</a> Proc Natl Acad Sci U S A. 81 (18), 5667-5671 (1984).</li><li>Resnick, R. J., Noreen, D., Munns, T. W., Perdue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+N6-methyladenosine+in+expression+of+Rous+sarcoma+virus+RNA:+analyses+utilizing+immunoglobulin+specific+for+N6-methyladenosine.">Role of N6-methyladenosine in expression of Rous sarcoma virus RNA: analyses utilizing immunoglobulin specific for N6-methyladenosine.</a> Prog Nucleic Acid Res Mol Biol. 29, 214-218 (1983).</li><li>Golovina, A. Y., 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=Method+for+site-specific+detection+of+m6A+nucleoside+presence+in+RNA+based+on+high-resolution+melting+(HRM)+analysis.">Method for site-specific detection of m6A nucleoside presence in RNA based on high-resolution melting (HRM) analysis.</a> Nucleic Acids Res. 42 (4), 27 (2014).</li><li>Dominissini, D., 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=Topology+of+the+human+and+mouse+m6A+RNA+methylomes+revealed+by+m6A-seq.">Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.</a> Nature. 485 (7397), 201-206 (2012).</li><li>Linder, B., 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=Single-nucleotide-resolution+mapping+of+m6A+and+m6Am+throughout+the+transcriptome.">Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.</a> Nat Methods. 12 (8), 767-772 (2015).</li><li>Fu, L., 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=Simultaneous+Quantification+of+Methylated+Cytidine+and+Adenosine+in+Cellular+and+Tissue+RNA+by+Nano-Flow+Liquid+Chromatography-Tandem+Mass+Spectrometry+Coupled+with+the+Stable+Isotope-Dilution+Method.">Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.</a> Anal Chem. 87 (15), 7653-7659 (2015).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondenaturing+agarose+gel+electrophoresis+of+RNA.">Nondenaturing agarose gel electrophoresis of RNA.</a> Cold Spring Harb Protoc. 2010 (6), 5445 (2010).</li><li>Wang, C. Y., 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=FTO+modulates+fibrogenic+responses+in+obstructive+nephropathy.">FTO modulates fibrogenic responses in obstructive nephropathy.</a> Sci Rep. 6, 18874 (2016).</li><li>Li, X., 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=Transcriptome-wide+mapping+reveals+reversible+and+dynamic+N-methyladenosine+methylome.">Transcriptome-wide mapping reveals reversible and dynamic N-methyladenosine methylome.</a> Nat Chem Biol. , (2016).</li><li>Sanschagrin, S., Yergeau, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+of+16S+ribosomal+RNA+gene+amplicons.">Next-generation sequencing of 16S ribosomal RNA gene amplicons.</a> J Vis Exp. (90), (2014).</li><li>Kukutla, P., Steritz, M., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+ribosomal+RNA+for+mosquito+gut+metagenomic+RNA-seq.">Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.</a> J Vis Exp. (74), (2013).</li><li>Mishima, 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=Immuno-Northern+Blotting:+Detection+of+RNA+Modifications+by+Using+Antibodies+against+Modified+Nucleosides.">Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.</a> PLoS One. 10 (11), 0143756 (2015).</li><li>Klungland, A., Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+RNA+modifications+in+disease.">Dynamic RNA modifications in disease.</a> Curr Opin Genet Dev. 26, 47-52 (2014).</li></ol>

저자는 공개 아무것도 없어.

Modifications of RNA have important roles in cellular function and physiology. The current understanding of the regulation, function, and homeostasis of these modifications is still being explored and expanded8. Therefore, a precise and gold-standard method to evaluate the modifications of RNA is needed. The modified northern blotting method provides precise quantification of RNA modifications and clear delineation of the modifications in diverse RNAs. Although the method requires at least 3 days, it can be standardized and can be used in various experimental designs. Moreover, with different antibodies, it can detect different RNA modifications27.
It is important to separate different RNAs when analyzing RNA modifications. Ribosomal RNA comprises a large portion of the total amount of RNA28,29. The results from analyzing RNA modifications only in total RNA will represent mostly the changes of rRNA. Methylation and other such modifications of rRNA could potentially mask the changes in other RNAs. With the procedure of gel separation, the modifications of mRNA and other small RNAs can be more accurately analyzed.
Transcriptome-wide mapping with m6A immunoprecipitation and sequencing provides detailed insight into the modification of each type of RNA22. It provides information on the specific RNAs and a resolution of around 80-120 bp. Although m6A-Seq can compare the modifications between different experimental conditions, the selection of proper standards and controls for such experiments is difficult18. Immunoprecipitation is difficult to reproduce, often giving significant variations amongst repeats. Moreover, m6A-Seq requires the fragmentation of RNA samples before immunoprecipitation and sequencing30. The fragmentation process could potentially induce undue influences on the original RNA modifications. If the experiment does not need the specific gene's information but requires different conditions for comparison, the current method provides better visualization and control for diverse experimental setups. 
Modification of RNA is an important step in regulating transcriptional control31. However, the homeostasis and the regulatory mechanisms of various RNA modifications under diverse physiological realms are still unclear. Using the present modified northern blotting method, different RNA modifications can be quantified and compared. Furthermore, the changes and regulations of RNA modifications can be investigated in greater detail. In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology.
The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. RNAs with some amount of degradation may yield good classical northern blotting results, but this could potentially have significant impact on the modified northern blotting results. The modifications of RNA in different tissues or cell lines could also vary significantly. It is important to test the suitable RNA sample loads for different tissues before performing the final experiments.
As blotting procedures have been traditionally named after Dr. Southern and different geographical directions, we propose the name "northeastern" blotting for the current technique.

Dynamic and reversible RNA modifications have important roles in RNA homeostasis. Four decades ago, N6-methyladenosine (m6A) modifications were found to be abundant in eukaryotic transcriptomes1. They have diverse functions in messenger RNA (mRNA), ribosomal RNA (rRNA), small nucleolar RNA, transfer RNA, and microRNA2. The m6A modifications of mRNA influence their splicing3, translation4, degradation5, and localization2. Moreover, they affect ribosome biogenesis and microRNA function6. The evolutionary conservation of m6A modifications of RNA is noted in unicellular bacteria to multi-cellular humans7. Delineation of the roles of m6A modifications is currently under extensive exploration. The efforts are expected to provide new insights into transcription control. Recent studies reveal that other chemical modifications of mRNA8 also play critical roles in RNA metabolism. 
Circadian rhythms9, stem cell pluripotency10, triglyceride metabolism4, fibrosis11, obesity12, and major depression13 are a few examples of processes where m6A modifications are known to control outcomes. Many circadian clock gene transcripts have m6A sites14. Modulation of m6A methylase or demethylase elicits circadian period changes15. Mettl3, an m6A transferase, is a regulator for stem cell pluripotency. A deficiency of Mettl3 leads to early embryonic lethality and aberrant lineage priming at the post-implantation stage10. A deficiency of fat mass- and obesity-associated (FTO), an m6A demethylase, in adipocytes affects fatty acid mobilization and body weight through posttranscriptional regulation of Angptl44. These studies reveal that m6A not only controls mRNA processing, but also plays critical roles in embryological development and patho-physiology. The function of m6A modifications holds implications for therapeutic considerations in the future.
Several methods are available to measure m6A modifications of RNA16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m6A in several RNAs19,20. Mass spectrometry is a sensitive tool for the detection of m6A modifications in RNA. However, the RNA needs to be excised by RNase into short fragments before analysis by mass spectrometry21. Methyl-RNA immunoprecipitation and sequencing (m6A-Seq)22 immunoprecipitates fragmented RNAs with m6A-specific antibodies and performs parallel RNA sequencing. This method generates transcriptome-wide m6A landscapes. High-resolution mapping of m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) further maps m6A modifications at a single-nucleotide resolution23. Both methods provide details of m6A modification across the whole transcriptome, with specific genes' information. However, quantifications and standardization in both methods are difficult if experiments require the comparison of multiple conditions. Moreover, fragmentations of RNA for m6A-Seq alter the original RNA structure, which may affect native m6A levels. To detect global m6A modifications of RNA and their changes under different experimental conditions, we report a method that employs a modified northern blotting protocol. This method resolves RNA by molecular weight, using gel electrophoresis18. This procedure provides better standardization and quantifications for experiments that involve multiple conditions or samples. It also provides specific m6A modification information for different RNAs, whether rRNA, mRNA, or microRNA.

<table><tbody><tr><td>RNaseZap solution</td><td>Ambion</td><td>AM9782</td><td>Protocol 1.1</td></tr><tr><td>TRI Reagent solution</td><td>Ambion</td><td>AM9738</td><td>Protocol 1.2.1.1</td></tr><tr><td>1-Bromo-3-chloropropane (BCP)</td><td>Sigma</td><td>B9673</td><td>Protocol 1.2.1.3</td></tr><tr><td>Ethanol</td><td>JTbaker</td><td>8006</td><td>Protocol 1.2.1.3</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>Protocol 1.2.1.3</td></tr><tr><td>GenElute mRNA Miniprep Kit</td><td>Sigma</td><td>MRN70</td><td>Protocol 1.2.2.1</td></tr><tr><td>Glycogen</td><td>Ambion</td><td>AM9510</td><td>Protocol 1.2.2.2</td></tr><tr><td>Sodium acetate</td><td>Fluka</td><td>71183</td><td>Protocol 1.2.2.2</td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9930</td><td>Protocol 1.2.2.6</td></tr><tr><td>DEPC</td><td>Sigma</td><td>D5758</td><td>Protocol 2.1.1</td></tr><tr><td>Agarose</td><td>JT Baker</td><td>A426</td><td>Protocol 2.1.2</td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td>Protocol 2.1.3</td></tr><tr><td>37% formaldehyde Solution</td><td>Sigma</td><td>F8775</td><td>Protocol 2.1.3</td></tr><tr><td>EDTA, Disodium Salt</td><td>JT Baker</td><td>8993</td><td>Protocol 2.1.3 10x MOPS buffer</td></tr><tr><td>formamide</td><td>Sigma</td><td>F7503</td><td>Protocol 2.2.1</td></tr><tr><td>RNA Millennium Marker</td><td>Ambion</td><td>AM7150</td><td>Protocol 2.2.2</td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>X328</td><td>Protocol 2.2.5</td></tr><tr><td>Ficoll PM400</td><td>GE Healthcare</td><td>17-0300-10</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>114391</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Xylene Cyanol FF</td><td>Sigma</td><td>X4126</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Sodium chloride dihydrate</td><td>JT Baker</td><td>3624</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Trisodium citrate</td><td>Sigma</td><td>S1804</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Hybond Blotting Paper</td><td>GE Healthcare</td><td>RPN6101M</td><td>Protocol 2.4.4</td></tr><tr><td>GE Hybond-N+ membrane</td><td>GE Healthcare</td><td>RPN303B</td><td>Protocol 2.4.6</td></tr><tr><td>Stratalinker UV Crosslinker 2400</td><td>Stratagene</td><td>400075</td><td>Protocol 3.1.2</td></tr><tr><td>Gel Catcher 1500</td><td>ANT Technology</td><td>Gel Catcher 1500</td><td>Protocol 3.1.5</td></tr><tr><td>Anti-m&lt;sup&gt;6&lt;/sup&gt;A (N&lt;sup&gt;6&lt;/sup&gt;-methyladenosine)</td><td>Synaptic Systems</td><td>202003</td><td>Protocol 3.2.3</td></tr><tr><td>Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td><td>GE Healthcare</td><td>NA934</td><td>Protocol 3.2.5</td></tr><tr><td>ChemiDoc MP System</td><td>BIO-RAD</td><td>1708280</td><td>Protocol 3.2.8</td></tr></tbody></table>

a method for measuring rna n6-methyladenosine modifications in cells and tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

정상 명암 주기성 단계 14 일 후에 야생형 마우스 상수 어둠 속에 넣었다. 간에서 RNA를 4 시간 간격으로 샘플링 및 수정 북부 블로 팅으로 연구 하였다. rRNAs, mRNA를, 작은 RNA를의 메틸화는 명확하게 (그림 2) 검출되었다. 상이한 주기성 시간 사이의 비교 (CT)을 정확하게 18S rRNA의 기준을 산출 할 수있다. rRNA의, mRNA를, 작은 RNA를에서 m 6 수준의 강력한 일중 진동이 있었다. rRNA의 proffered 의해 간섭을 방지하기 위해, 폴리아 데 닐화 RNA를 단계 1.2.2와 같이 정제 될 수있다. 정제 후, rRNA의 크게 다른 RNA를 (도 3)의보다 나은 시각화를 허용하도록 제거 될 수있다. <img alt="그림 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt;도 1 : 오 이송 유닛 조립. 멤브레인에 블 롯팅을위한 RNA를 모세관 반송 유니트가 도시되어있다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> 그림 2 : C57BL / 6J 마우스의 m 6 수준의 활동 일주기. 이 그림은 m 6 야생형 생쥐의 간에서 총 RNA의 얼룩은 4 시간 간격으로 정상 명암 단계 14 일 이후에 어두운 어두운 단계의 첫 번째 날에 희생 보여줍니다. 정량화는 6 m 18S와 28S rRNA의 사이의 화상 농도의 차이를 사용하여 수행 하였다. m 개의 6 풍요 로움은 맨 아래에있는 포름 알데히드 겔에서 18S rRNA의 대역으로 표준화되었다.등 = &quot;_ 빈&quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> 그림 3 : 또는 rRNA의없는 m 6의 말. 대표 m는 총 RNA와 야생형 생쥐의 간에서 폴리아 데 닐화 RNA의 6 롯이 표시됩니다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1 </td><td colspan="3"> 10 배 MOPS 완충액 (pH 7.0, 빛으로부터 보호) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> MOPS </td><td> 41.85 </td><td> 지 </td><td> 0.2 M </td></tr><tr><td></td><td colspan="3"> 아세트산 나트륨 </td><td> 4.1 </td><td> 지 </td><td>0.05 M </td></tr><tr><td></td><td colspan="3"> EDTA,이 나트륨 소금 </td><td> 3.7 </td><td> 지 </td><td> 0.01 M </td></tr><tr><td></td><td colspan="3"> DEPC 물 </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 합계 </td><td> 1 </td><td> 엘 </td><td></td></tr><tr><td></td><td colspan="3"> 실온에서 교반 </td><td></td><td></td><td></td></tr><tr><td> 이 </td><td colspan="3"> 20 배의 SSC 완충액 (pH 7.0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 염화나트륨 </td><td> 175.3 </td><td> 지 </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> 트리 소듐 시트 레이트 </td><td> 88.3 </td><td> 지 </td><td> 0.3 M </td></tr><tr><td></td><td colspan="3"> DEPC 물 </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 합계 </td><td> 1 </td><td> 엘 </td><td></td&gt; </tr><tr><td></td><td colspan="3"> 실온에서 교반 한 후, 오토 클레이브 </td><td></td><td></td><td></td></tr><tr><td> 삼 </td><td colspan="3"> 추적 염료 </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10 배 MOPS 버퍼 </td><td> (500) </td><td> μL </td><td> 1 배 </td></tr><tr><td></td><td colspan="3"> 피콜 (400) </td><td> 0.75 </td><td> 지 </td><td> 15 % </td></tr><tr><td></td><td colspan="3"> 블루 브로 모 페놀 </td><td> 0.01 </td><td> 지 </td><td> 0.2 % </td></tr><tr><td></td><td colspan="3"> 크실렌 Cyanol </td><td> 0.01 </td><td> 지 </td><td> 0.2 % </td></tr><tr><td></td><td colspan="3"> DEPC 물 </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 합계 </td><td> (5) </td><td> mL의 </td><td></td></tr><tr><td></td><td colspan="3"> 스토어 -20 ° C에서 </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC 물 </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 1 비율로 희석 : DEPC의 1,000 DDH 2 O에 </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 실온에서 밤새 교반 한 후, 오토 클레이브 </td><td></td><td></td><td></td></tr><tr><td> (5) </td><td colspan="3"> 샘플 버퍼 (빛으로부터 보호) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10 배 MOPS 버퍼 </td><td> (200) </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> 37 % 포름 알데히드 </td><td> (270) </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> 포름 아미드 </td><td> (660) </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> 스토어 -20 ° C에서 </td><td></td><td></td><td></td></tr></tboDY&gt; </table> 표 1 : 버퍼 및 솔루션을 제공합니다.

Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

RNA에 N 6 -methyladenosine (m 6 A) 변형을 측정하기위한 수정 북부 블로 팅 방법을 설명한다. 현재의 방법은 여러 가지 실험 설계에 따라 다양한 RNA를에서 수정 및 컨트롤을 감지 할 수 있습니다.

RNA 측정 방법<em&gt; N</em<sup&gt; 6</sup세포와 조직에서&gt; -methyladenosine 수정

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

a-method-for-measuring-rna-n6-methyladenosine-modifications-cells

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

10.8K 조회수.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  RNA에 N 6 -methyladenosine (m 6 A) 변형을 측정하기위한 수정 북부 블로 팅 방법을 설명한다. 현재의 방법은 여러 가지 실험 설계에 따라 다양한 RNA를에서 수정 및 컨트롤을 감지 할 수 있습니다. 

비디오: A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

TRNA Isopentenyl 전이 효소 활동을 감지 하는 체 외에서 분석 결과

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

연구

생화학

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA 메틸 트랜스퍼라제 활성 분석을 위한 항체 무첨가 분석

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

유전학

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

작은 RNA-seq 개선: 2'-O-메틸 RNA의 더 적은 편견 및 더 나은 검출

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

메틸화 RNA 면역 침전 분석연구 m5C 변형에 대한 애비독증

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

핵자기공명분광법에 의한 아르기닌 메틸롬 탐구

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

암 연구학

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

HPLC-MS/MS를 사용하여 뉴클레오시드/뉴클레오티드 함량 측정을 위한 식물 샘플 준비

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

RRNA 2'-O-메틸화의 DNAzyme 의존분석

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

발생학

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

질량 분광법을 사용하여 단일 뉴런의 RNA 변형 특성화

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

RNA 측정 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

TRNA Isopentenyl 전이 효소 활동을 감지 하는 체 외에서 분석 결과

RNA 메틸 트랜스퍼라제 활성 분석을 위한 항체 무첨가 분석

작은 RNA-seq 개선: 2'-O-메틸 RNA의 더 적은 편견 및 더 나은 검출

메틸화 RNA 면역 침전 분석연구 m⁵C 변형에 대한 애비독증

2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

2D-HELS MS 염기서열분석(2D-HELS MS SEQ): 뉴클레오티드 변형이 다른 RNA 혼합물의 직접 및 새로운 염기서열분석을 위한 일반적인 LC-MS 기반 방법

핵자기공명분광법에 의한 아르기닌 메틸롬 탐구

HPLC-MS/MS를 사용하여 뉴클레오시드/뉴클레오티드 함량 측정을 위한 식물 샘플 준비

RRNA 2'-O-메틸화의 DNAzyme 의존분석

질량 분광법을 사용하여 단일 뉴런의 RNA 변형 특성화