이 작품은 싱가포르 NRF의 교제 (NRF-2011NRF - NRFF001-025)뿐만 아니라 CBRG (NMRC / CBRG가 / 0070 / 2014)에 의해 투자되었다.

참고 :이 연구의 맥락에서 관심의 RNA는 AR 3&#39;UTR의 단편이다. 비오틴 표지 RNA 1. 준비 <ol><li> RNA를 얻으려면, 상용 키트를 사용하여 제조 업체의 지침 (11)는 다음의 시험 관내 전사 다음 PCR에 의해 T7-AR-올리고을 증폭. 참고 : PCR 프라이머는 표 4에 나열되어 있습니다 : T7-AR-F와 T7-AR-R을. </li><li> 비특이적 결합 제어를 들어, 시험 관내 전사 하였다 psiCHECK -2- 플라스미드로부터 PCR에 의해 반딧불 루시페라아제 (FL) DNA의 부분 서열을 증폭. 참고 : PCR 프라이머는 표 4에 나열되어 있습니다 hluc (+) - T7 프로브 F 및 hluc (+) - 프로브-R 표 4 PCR 증폭을위한 템플릿과 프라이머 시퀀스... </li><li> 50 ㎕의 PCR 반응에서, 50 ng의의 DNA (올리고 또는 플라스미드)의 4 μL의 dNTPs (각 2.5 mM의 dNTP), 5 ㎕의 10 배 반응 완충액 1 ㎕를 2.5 U / &amp; # 믹스(181), 리터 DNA 중합 효소, 2 μL 10 μm의 정방향 프라이머, 2 μL 10 μm의 역방향 프라이머, 및 뉴 클레아없는 물. <ol><li> 다음 프로그램을 사용하여 열 순환기 PCR 증폭을 실행. STEP1 : 2 분, 95 ℃ 1주기; STEP2 : 30 초 동안 95 ° C의 35주기, 30 초 동안 55 ° C 및 30 초 (AR) 또는 60 초 (FL) 72 ° C; STEP3 : 10 분 동안 72 ℃에서 1주기. </li></ol></li><li> 제조업체의 지침 11 다음 시험 관내 전사 키트를 사용하여 바이오틴의 RNA를 합성. 이 실험에서는, RNA 합성 템플릿으로 PCR 제품을 사용한다. 20 ㎕의 시험 관내 전사 반응에 200 ng를 PCR 증폭 된 DNA, 각 NTP 2 μL, 1 ㎕의 10 mM이 비오틴 CTP-2 배 μL 반응 완충액, 2 μL의 T7 RNA 폴리머 라제를 혼합한다. 4 시간 동안 37 ℃에서 혼합물을 인큐베이션. </li><li> 37 &amp; #에서 시험 관내 전사, 1 μL 2 U / μL DNase의와 치료 반응 혼합물에 따라(176), C 15 분 주형 DNA를 분해합니다. 마지막으로, 이후의 정제하여 60 ㎕의 총 부피로 오티 닐화 RNA 희석. </li><li> 제조업체의 지침 12 다음 정화 열을 사용하여 염 및 비법 인 세포핵을 제거하기 위해 비오틴 RNA를 정화. 다음 풀다운 분석을 위해 -80 ° C에서 RNA 농도 및 저장을 측정합니다. </li><li> 2 분, 2 분 동안 얼음에 진정 90 ° C에서 RNA 단백질 상호 작용, 열 50 μL 비오틴 RNA (50 PM)에 대한 관심의 RNA에 대한 적절한 이차 구조를 보장하기 위해, 다음 50 μl의 2 배 RNA 구조 버퍼 (표와 공급 3), 13, 14 및 30 분 동안 실온에서 접는 RNA 허용한다. </li></ol> RNA 접합 비즈 2. 준비 참고 : 자기 구슬과 상등액의 분리 자기 스탠드를 사용합니다. <ol><li> 간단한 vorte하여 원래 바이알의 스트렙 타비 딘 - 코팅 된 자성 비드를 재현 탁싱은 제조업체의 지침에 따라. 1.5 ML 튜브에 비즈를 재현 탁 150 μl를 전송합니다. 1 분 동안 자석에 튜브를 놓습니다. 폐기 뜨는. 비드 펠렛을 유지합니다. 참고 : RNA 풀다운 분석에서, 원래 유리 병에서 재현 탁 구슬을 분취 때, MS 다음에 분석을 위해 서부 블롯 다음에 분석을 위해 50 μl의 구슬, 200 μl의 구슬을 사용합니다. </li><li> 다시 현탁 제조자의 권장 1X W 및 B의 1 ml의 비드 펠렛 (결합 및 세척) 완충액 (표 3)에 의해 회 비드 워시 회전기에 실온에서 5 분 동안 튜브를 회전 하였다. 폐기 뜨는. 비드 펠렛을 유지합니다. </li><li> 400 ㎕의 완충액 A의 완충액 A로 비드 펠렛을 재 - 현탁하여마다, 구슬의 RNase 불 활성화 회 비드 씻어 0.1 M NaOH를 0.05 M의 NaCl (표 3)를 함유하는 알칼리성 용액이다. 회 전자에서 실온에서 2 분 동안 튜브를 돌린다. 폐기 뜨는. 비드 펠렛을 유지합니다. </li> &lt;리&gt; 회전기 실온에서 2 분 동안 튜브를 회전 뒤에 다시 현탁 400 ㎕의 완충액 B로 비드 펠렛 (표 3)에 의해 회 비드 씻어 비즈마다 NaOH를 제거한다. 폐기 뜨는. 비드 펠렛을 유지합니다. </li><li> 300 ㎕의 2 배 W &amp; B 버퍼를 Resuspend 비드 펠렛, 분할 구슬 똑같이 세 1.5 ml의 튜브 (튜브 당 100 μl의 구슬)로 전송합니다. 100 μL의 RNase없는 물과 비드 함유 공급 튜브 100 μL는 FL의 RNA 각각 AR 3&#39;UTR RNA 바이오틴 100 μL를 비 오티 닐화. </li><li> 회전을 실온에서 1 시간 동안 각 비드 혼합물을 인큐베이션. 1 분 동안 자석에 튜브를 놓습니다. 모든 상층 액을 버린다. 비드 펠렛을 유지합니다. </li><li> 400 ㎕의 1X W 및 B 버퍼 각각 비드 펠렛을 다시 현탁 회마다 비드를 세척 회전기에 실온에서 5 분 동안 튜브를 회전 하였다. </li><li> 1 분 동안 자석에 튜브를 놓습니다. 모든 상층 액을 버린다. 각 비드 PELLE를 재현 탁50 μl의 핵 분리 버퍼 (표 3)와 t. </li></ol> 3. 지방 전구 세포의 분리 및 지방 생성 유도 <ol><li> 이전 서적 5에 기재된 바와 같이, 견갑골 갈색 지방 패드로부터 전구 지방 세포 (C57BL6 야생형 마우스, 3 주)를 해부. </li><li> 성장과 체외에서 전구 지방 세포 (5) 차별화. 세포는 4~5일 분화 개시 후 다운 스트림 응용 프로그램에 대한 준비가되어 있습니다. </li></ol> 차 지방 세포에서 세포 용 해물 4. ​​준비 주 : 파쇄 제제의 모든 절차를 확인 얼음에 만들어진 모든 버퍼 얼음에 냉각된다. 얼음 사전 냉각 다운스 유리 균질. <ol><li> 성장과 15cm 요리 (5) (3.2 절 참조) 전구 지방 세포를 분화. 각각의 요리는 하나의 RNA 풀다운 분석을위한 충분한 세포를 제공한다. 일 4 분화 폐기 세포 배양액 일 5, 세탁에서 CE한 번 1X PBS와 LLS. </li><li> RT에서 5 분 200 XG에서 원심 분리하여 50 ML 튜브에 세포 및 펠렛 세포를 수집 긁어, 세포를 수확 세포에 10 ~ 15 ㎖의 1X PBS를 추가합니다. 1 ML의 피펫을 이용하여 상층 액을 버린다. </li><li> 재현 탁 셀이 용액에 펠릿 저장성 완충액 (표 3), 및 15 분 동안 얼음 위에서 세포를 배양한다. 전송 15 ml의 다운스 유리 균질에 세포 및 기계적으로 얼음에 20 스트로크와 전단 세포. </li><li> 4 ° C에서 15 분 동안 3,300 XG에 원심 분리하여 펠렛은 세포 핵. 더 사용하기 위해 얼음에 핵 펠렛을 유지 (섹션 4.6 참조). 1.5 ml의 튜브에 뜨는을 전송, 4 ℃에서 15 분 동안 20,000 XG에서 최종 150 mm의 농도와 스핀을 얻을 수 뜨는하는 3M의 KCl을 추가합니다. </li><li> 원심 분리 후, 조심스럽게 1 ml를 피펫을 사용하여 상단 lipidlayeron 제거하고 세포질 세포 용 해물로 뜨는를 수집합니다. </li><li> 핵 펠렛을 재현 탁 1 ml의 핵 분리 버퍼 (4.4 절 참조). 티를 ransfer의 1 ML의 피펫을 사용하여 15 ml의 다운스 유리 균질에 핵, 기계적으로 100 스트로크와 얼음에 전단 핵. 4 ° C에서 15 분 동안 20,000 XG에서 원심 분리하여 핵 파편 펠렛, 핵 세포 용 해물로 뜨는를 수집합니다. </li><li> 어느 쪽의 핵 및 세포질 세포 용 해물을 결합, 또는 다운 스트림 풀다운 응용 프로그램에 대해 개별적으로 각 부분을 사용합니다. 데모 실험에서, 세포 용 해물이 두 분획 1의 부피비로 혼합 하였다 1. </li><li> 0.5 U / μL의 최종 농도를 얻기 위해,이 분획을 세포 용 해물의 RNase 억제제를 추가한다. 웨스턴 블롯 (15)의 입력 컨트롤로 세포 용 해물의 옆 100 μl를 설정합니다. </li></ol> 5. 바인딩 및 RBP의 용출 <ol><li> 분할 셀 세 동일한 분취 량에 해물 (1.5 ML 튜브에 1 ㎖의 세포 용 해물), 빈과 부화는 FL RNA 접합 및 AR 3&#39;UTR RNA 접합 비즈는 4 °에서 3 시간 동안 각각 (섹션 2.8 참조) 기음회전. </li><li> 1 분 동안 자석에 튜브를 놓습니다. RNA의 완전성을 확인하기 위해 상층 액에서 RNA 분리에 이어 1 ml를 피펫을 이용하여 상층 액을 수집합니다. 제조자의 지시 (표 2) 다음 산 구 아니 디늄 티오 시아 네이트 - 페놀 용액을 이용하여 RNA를 분리. 리보좀 RNA의 28S 및 18S 서브 유니트를 분석해서 RNA의 intactness 평가. 얼음에 비드 펠렛을 유지합니다. </li><li> 회 전자에서 실온에서 2 분 동안 튜브를 회전이어서 40 U의 RNase 억제제 및 0.25 % IGEPAL를 함유 한 용액의 핵 차단 완충액으로 각 비드 펠렛을 재현 탁하여 자성 비드 여섯 번마다 세척 하였다. 자기 구슬과 상층 액의 분리를위한 자기 스탠드를 사용합니다. 모든 상층 액을 버린다. 얼음에 비드 펠렛을 유지합니다. </li><li> 웨스턴 블로 팅을위한 비즈에서 RNA 단백질 복합체를 용출 다음 단계를 사용하십시오. 먼저, 75 μL 핵 분리 버퍼의 각 비드 펠렛을 재현 탁; 다음, 25 ㎕의 4 배 웨스턴 블롯 로딩 버피를 추가FER은 (SDS 함유) 100 μl의 총 부피를 얻었다. 마지막 5 분 동안 95 ℃에서 100 μL이 용액에 비드를 끓인다. 참고 : 사용이 단계는 MS에 대한 비즈에서 RNA 단백질 복합체를 용출 다음과 같습니다. STEP1 : 100 μL 용출 버퍼 (표 3)의 각 비드 펠렛을 재현 탁; STEP2는 : 회전 실온에서 2 ~ 3 시간 동안 비드 샘플을 품어; STEP3 : 원심 분리기와 단백질 함유 상층 액을 수집; STEP4는 : MS에 제출하기 전에 20 ~ 30 μL에 단백질 솔루션을 집중한다. </li><li> 원심 분리기는 각각 100 4 ° C에서 30 초 동안 12,000 XG에 비드 샘플의 μL, 그리고 상층 액을 수집합니다. 웨스턴 블롯 분석 (섹션 6.1 및 6.2 참조) 나누어지는 15 μl의 상층 액. 하룻밤 (1000 희석 1) 희석 허 마우스 단일 클론 항체로 인한 막 프로브. </li></ol> RBP의 검증 6. 웨스턴 블로 팅 <ol><li> 표준 기술을 이용하여 SDS-PAGE에 의해 분리 RBPs. </li><li> 우리를 실시선미 관심의 RNA와 관련된 RBPs에 대한 프로빙에 의해 표준 기술을 사용하여 블로 팅. 주 :이 데모 실험에서 허 마우스 모노클로 날 항체 허 단백질의 면역 검출에 사용되었다. </li><li> 희석 허 항체 생성 된 막 품어 : w / V의 탈지 분유, 1 배 TBS 5 % (1 천 희석) 0.1 % 트윈 - 20 (4)에서 하룻밤, 부드러운 진탕 C를 °. 1X TBST로 막 세 번 씻으십시오. 1 시간 동안 실온에서 (항 - 마우스 IgG-HRP 염소) 이차 항체와 함께 막을 인큐베이션. 워시 막. 개발 및 기록 신호. </li></ol>

<ol>
	<li>Huot, M. &. #. 2. 0. 1. ;., 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+Sam68+STAR+RNA-binding+protein+regulates+mTOR+alternative+splicing+during+adipogenesis.">The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.</a> Mol Cell. 46 (2), 187-199 (2012).</li><li>Sun, 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=Long+noncoding+RNAs+regulate+adipogenesis.">Long noncoding RNAs regulate adipogenesis.</a> Proc Natl Acad Sci U S A. 110 (9), 3387-3392 (2013).</li><li>Engreitz, 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=The+Xist+lncRNA+exploits+three-dimensional+genome+architecture+to+spread+across+the+X-chromosome.">The Xist lncRNA exploits three-dimensional genome architecture to spread across the X-chromosome.</a> Science. 341 (6147), 1237973 (2013).</li><li>Zhao, X. Y., Li, S., Wang, G. X., Yu, Q., Lin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long+noncoding+RNA+transcriptional+regulatory+circuit+drives+thermogenic+adipocyte+differentiation.">A long noncoding RNA transcriptional regulatory circuit drives thermogenic adipocyte differentiation.</a> Mol Cell. 55, 372-382 (2014).</li><li>Alvarez-Dominguez, J. R., 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=De+novo+reconstruction+of+adipose+tissue+transcriptomes+reveals+long+non-coding+RNA+regulators+of+brown+adipocyte+development.">De novo reconstruction of adipose tissue transcriptomes reveals long non-coding RNA regulators of brown adipocyte development.</a> Cell Metab. 21, 764-776 (2015).</li><li>Adams, D. J., Beveridge, D. J., van der Weyden, L., Mangs, H., Leedman, P. J., Morris, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HADHB,+HuR,+and+CP1+bind+to+the+distal+3-untranslated+region+of+human+renin+mRNA+and+differentially+modulate+renin+expression.">HADHB, HuR, and CP1 bind to the distal 3-untranslated region of human renin mRNA and differentially modulate renin expression.</a> J Biol Chem. 278 (45), 44894-44903 (2003).</li><li>Yeap, B. 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=Novel+binding+of+HuR+and+poly(C)-binding+protein+to+a+conserved+UC-rich+motif+within+the+3&#180;+untranslated+region+of+the+androgen+receptor+messenger+RNA.">Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 3&#180; untranslated region of the androgen receptor messenger RNA.</a> J Biol Chem. 277, 27183-27192 (2002).</li><li>K&#246;nig, J., Zarnack, K., Luscombe, N. M., Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-RNA+interactions:+new+genomic+technologies+and+perspectives.">Protein-RNA interactions: new genomic technologies and perspectives.</a> Nat Rev Genet. 13, 77-83 (2012).</li><li>Ferr&#232;, F., Colantoni, A., Helmer-Citterich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+protein-lncRNA+interaction.">Revealing protein-lncRNA interaction.</a> Briefings in Bioinformatics. , 1-11 (2015).</li><li>McHugh, C. A., Russell, P., Guttman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+comprehensive+experimental+identification+of+RNA-protein+interactions.">Methods for comprehensive experimental identification of RNA-protein interactions.</a> Genome Biol. 15 (1), 203 (2014).</li><li>Lee, Y. 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=AU-rich+element-binding+protein+negatively+regulates+CCAAT+enhancer+binding+protein+mRNA+stability+during+long+term+synaptic+plasticity+in+Aplysia.">AU-rich element-binding protein negatively regulates CCAAT enhancer binding protein mRNA stability during long term synaptic plasticity in Aplysia.</a> Proc Natl Acad Sci U S A. 109 (38), 15520-15525 (2012).</li><li>Hermann, M., Cermak, T., Voytas, D. F., Pelczar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+genome+engineering+using+designer+nucleases.">Mouse genome engineering using designer nucleases.</a> J Vis Exp. (86), (2014).</li><li>Tsai, M. C., 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=Long+Noncoding+RNA+as+Modular+Scaffold+of+Histone+Modification+Complexes.">Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes.</a> Science. 329 (5992), 689-693 (2010).</li><li>Wan, 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=Landscape+and+variation+of+RNA+secondary+structure+across+the+human+transcriptome.">Landscape and variation of RNA secondary structure across the human transcriptome.</a> Nature. 505, 706-709 (2014).</li><li>Li, H., 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=Identification+of+mRNA+binding+proteins+that+regulate+the+stability+of+LDL+receptor+mRNA+through+AU+rich+elements.">Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU rich elements.</a> J Lipid Res. 50 (5), 820-831 (2009).</li><li>McHugh, C. A., 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+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521 (7551), 232-236 (2015).</li><li>Chu, C., 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=Systematic+Discovery+of+Xist+RNA+Binding+Proteins.">Systematic Discovery of Xist RNA Binding Proteins.</a> Cell. 161 (2), 404-416 (2015).</li><li>Chu, C., Spitale, R. C., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+to+probe+functions+and+mechanisms+of+long+noncoding+RNAs.">Technologies to probe functions and mechanisms of long noncoding RNAs.</a> Nat Struct Mol Biol. 22 (1), 29-35 (2015).</li><li>Zhao, J., 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=Genome-wide+identification+of+Polycomb-associated+RNAs+by+RIP-seq.">Genome-wide identification of Polycomb-associated RNAs by RIP-seq.</a> Mol Cell. 40 (6), 939-953 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

lncRNAs 및 RBPs 그러나 이러한 분자의 분자 메커니즘을 제대로 이해, 건강과 질병에 중요한 역할을합니다. lncRNA 분자와 상호 작용하는 단백질의 식별 규제 메커니즘을 해명을 향한 중요한 단계입니다. RNA의 풀다운 분석 시스템에 의한 RBPs의 보충은 세포 용 해물로부터 단백질을 선택적 스트렙 자성 비드에 결합 된 비오틴 RNA 베이트를 사용하여 추출 될 수 있도록 복잡한 상호 작용의 인 용액 캡쳐에 ...

A correction to the author list was made to: <a href="http://www.jove.com/video/54207/detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte">Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture</a>.
The author list has been updated from:
Qianfan Bai1*, Zhiqiang Bai1*, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
*These authors contributed equally
to:
Qianfan Bai1*, Zhiqiang Bai1*, Shaohai Xu3, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
3Division of Bioengineering, School of Chemical &#38; Biomedical Engineering, Nanyang Technological University
*These authors contributed equally

A correction to the author list was made to: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

Erratum: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RBPs 세포의 이중 또는 단일 가닥 RNA에 결합하여 RNA - 단백질 복합체 형성에 참여하는 단백질이다. RBPs는 mRNA의 긴 비 암호화 RNA를 (lncRNAs)를 포함하는 RNA 종의 다양한 결합 및 전사 후 수준에서 자신의 영향력을 행사할 수있다. 두 RBPs 및 lncRNAs은 지방 개발 등 새로운 규제를 신흥 1,2,3 작동된다. 이 특정 RNA 분자와 하나 이상의 RBPs 사이의 상호 작용을 검출하는 것이 필요는 RBP- 메커니즘 및 세포질 통로의 lncRNA 매개 규제를 이해하고, 때로는 RNA의 단백질 파트너의 전체 스펙트럼을 식별 할 성적 증명서. 그러나, 실험에 의한 지방 세포에서 지질의 높은 함량에 어려울 수있다. 여기에 설명 된 RNA 풀다운 프로토콜 4,5 차 배양 지방 세포의 용 해물에서 특정 RNA의 단백질 파트너를 검색하기 위해 사용될 수있다. 이 프로토에 대한 이론적 근거다음 COL 요약된다. RNA를 미끼는 DNA 주형으로부터 전사 된 시험관에, 비오틴 - 표지 된 스트렙 타비 딘 - 코팅 된 자성 비드 (4)과 공액. RNA를 미끼이어서 자기 스탠드에서 내려 오게되는 RNA 단백질 복합체의 형성을 허용하는 세포 용 해물과 배양된다. 구체적으로는, RNA 풀다운 프로토콜은 후술 허 단백질 일차 배양 theadipocytelysate에서의 mRNA의 3&#39;UTR 6,7 결합 보편적 RBP 발현을 검색 미끼 AR 3&#39;UTR로부터 RNA 단편을 사용한다. 이 분석의 결합 특이성을 테스트하려면, 비 관련 RNAas 아니라 빈 스트렙 타비 딘 비드 제어 포함되어 있습니다. 이 프로토콜은 특정 RBP의 포착을 확인하거나 캡처 RBPs 각각 8의 전체 레퍼토리를 식별하는 웨스턴 블 롯팅 또는 질량 분석기 (MS)와 호환된다. 몇 가지 기술은 RNA 단백질 상호 작용을 연구하는 데 사용할 수 있습니다에스. 주어진 RBP, RIP (RNA의 면역)와 CLIP (UV 가교 및 면역)에 구속 RNA를 공개하기 위해하는 것은 적용 할 수 있습니다. 반대로, 소정의 RNA의 단백질 파트너를 식별하기 위해, RNA 풀다운는 처프 (RNA 정제하여 염색질 분리) CHART (RNA 타겟 캡처 혼성화 분석)와 RAP (RNA 안티센스 정제)에 적용될 수있다. 나중에 사람과 비교하여, RNA 풀다운 기술은 설정하는 작은 노력이 필요합니다. 생체 외 및 생체 내에서 단백질을 포착하기 위해 사용될 수있다. 그 시험관 대응보다 기술적으로 더 도전 RNA 풀다운,의 생체 방법은 세포에서 가교에 의해 RNA 단백질 상호 작용을 유지, 세포로부터 관심의 앱 타머 - 태그 RNA를 캡처하고,이어서 바인딩 RBPs를 감지합니다. RNA 풀다운 낮은 풍부한 RBPs 농축 및 분리 및 세포 조절을 제어 9,10 다양한 기능적 역할이 RNA 단백질 복합체를 식별하기 위해 사용될 수있다 </s&gt;입니다.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:499px;">Major materials used in this study.</td>
			<td style="width:177px;"></td>
			<td style="width:109px;"></td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEGAscript kit</td>
			<td>&#160;Ambion</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin-14-CTP&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucAway spin columns&#160;</td>
			<td>Ambion</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HuR (3A2) mouse monoclonal antibody (sc-5261)&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG-HRP(sc-2005)&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hypure Molecular Biology Grade Water (nuclease-free)&#160;</td>
			<td>HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (1&#215;PBS)</td>
			<td>&#160;HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase inhibitor&#160;</td>
			<td>Bioline</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pfu Turbo DNA polymerase&#160;</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIsure</td>
			<td>&#160;Bioline</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Major equipment used in this study.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend Micro 21R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend X1R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Rad T100 thermal cycler&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop 2000&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BOECO rotator Multi Bio RS-24&#160;</td>
			<td>BOECO,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein gel electrophoresis and blotting apparatus&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2 magnet&#160;</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture

RNA 결합 단백질 (RBPs)은 지방의 개발 및 기능의 규제 계층으로 부상하고있다. RBPs 대상의 mRNA의 안정성 및 병진 효율에 영향을 미치는하여 전사 후 수준에서 유전자 발현을 조절하는 중요한 역할을한다. RNA 풀다운 기술은 널리기구 RBPs &#39;아니라 긴 비 코딩 RNA를&#39;(lncRNAs) 함수를 기초 해명 할 필요가 RNA - 단백질 상호 작용을 연구하기 위해 사용되었다. 그러나, 지방 세포에서 높은 지질 풍요 로움이 실험 수행에 기술적 인 도전을 포즈. 다음은 자세한 RNA 풀다운 프로토콜은 기본 지방 세포 배양에 최적화되어 있습니다. 안드로겐 수용체의 (AR) 3 &#39;번역되지 않은 지역 지방 세포 lystate에서의 RBP 파트너, 허 단백질을 검색하는 방법을 보여주기 위해 예를 들어 사용되는 아데 닐 - uridylate 풍부한 elementwas를 포함 (3&#39;UTR)에서 RNA 조각. 여기에 설명 된 방법의 상호 작용을 검출하기 위해 적용될 수있다RBPs 및 비 암호화 RNA를,뿐만 아니라 사이 RBPs 및 코딩 RNA를.

데모 실험에서, AR RNA 단편은 결합 단백질 허 캡처 미끼로서 사용 하였다. 모두 허 단백질 비 관련있는 플로리다 RNA 미끼 ​​및 접합 빈 스트렙 타비 딘 비드의 분취 량은 부정적인 컨트롤을 제공합니다. RNA - 단백질 상호 작용은 핵 또는 세포질 중 발생할 수 있으며,이 풀다운 프로토콜 또는 총 분획 (핵 또는 세포질) 세포 용 해물 중 하나에 적용될 수있다. 웨스턴 블로 팅?...

Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA 풀다운 프로토콜은 RNA 결합 단백질 (RBPs) 및 비 암호화뿐만 아니라 코딩 RNA를 사이의 상호 작용의 검출을 위해 여기에 최적화되어 있습니다. 안드로겐 수용체 (AR)에서 RNA 조각은 차 갈색 지방 세포의 lystate로부터 RBP를 검색하는 방법을 보여주기 위해 예로서 사용되었다.

RNA 결합 단백질의 검출에 의해<em&gt; 체외</em지방 세포 문화&gt; RNA 풀다운

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte

Research

JoVE Journal

Biology

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

23.5K 조회수.  Duke-NUS Medical School.  RNA 풀다운 프로토콜은 RNA 결합 단백질 (RBPs) 및 비 암호화뿐만 아니라 코딩 RNA를 사이의 상호 작용의 검출을 위해 여기에 최적화되어 있습니다. 안드로겐 수용체 (AR)에서 RNA 조각은 차 갈색 지방 세포의 lystate로부터 RBP를 검색하는 방법을 보여주기 위해 예로서 사용되었다. 

비디오: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR - 클립 - RNA 단백질의 바인딩 Transcriptome 전체 바인딩 사이트를 식별하는 방법

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

연구

생물학

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

시토크롬의 세포질 Microinjection 의한 Apoptosis의 활성화 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

풀다운 Calmodulin 결합 단백질의

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

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 ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

천연 단백질에서 아밀로이드의 빠른 생성<em&gt; 체외</em

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

작은 RNA 종의 북부 오 탐지의 향상된<em&gt; 초파리 Melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

인간 성숙한 지방세포의 고립과 문화는 막 성숙한 지방 세포 골재 배양 (MAAC)를 사용하여

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...