RNA 정제에 의한 염색질 절연 (처프)

Biology
JoVE Journal
Biology
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Summary

처프 긴 noncoding RNAs의 게놈에 바인딩 사이트 (lncRNAs)를 매핑하는 소설 및 신속한 기술입니다. 방법은 lncRNA 바인딩된 게놈 사이트의 열거를 허용하는 안티 센스가 원래의 oligonucleotides의 특이성을 이용합니다.

Cite this Article

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Chu, C., Quinn, J., Chang, H. Y. Chromatin Isolation by RNA Purification (ChIRP). J. Vis. Exp. (61), e3912, doi:10.3791/3912 (2012).

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Abstract

긴 noncoding의 RNAs에는 투여 보상, 각인, 그리고 발달 유전자 발현 1,2,3,4,5,6,7 같은 중요한 생물 학적 과정 동안 염색질 상태 중 핵심 규제입니다. mediates 히스톤 H3 라이신 27 trimethylation (H3K27me3)가 유전자에 특정한에서 염색질 상태를 관리하는 수많은 lncRNAs에 대한 광범위한 역할을 제시하는 등 Polycomb 억압 단지 2 (PRC2)와 같은 특정 염색질의 수정 단지와 협회의 lncRNAs 수천명의 최근 발견 패션 8,9. 일부 lncRNAs은 인근의 유전자에있는 CIS에서 일할 것으로 생각되고 있지만, 다른 lncRNAs가 먼에 위치한 유전자를 조절하는 트랜스으로 작동합니다. 예를 들어, Drosophila lncRNAs roX1과 남성 세포 X의 염색체에 roX2 구속 수많은 지역 및이 복용량 보상 10,11 위해 중요합니다. 그러나 그들의 구속력이 사이트의 정확한 위치는 높은 해상도로 알려져 있지 않습니다. 마찬가지로, 인간 lncRNA HOTAIR은 H에 PRC2 인 영향을 미칠 수유전자 게놈 차원 3,12,13,하지만 어떻게 특이성이 이루어지면의 undreds가 불분명합니다. LncRNAs 또한 여러 단백질 단지의 어셈블리를 채용한 모듈형 공사장 공중 발판이 될 수 있습니다. 클래식 트랜스 연기 RNA 비계 14 복잡한 telomerase를위한 템플릿 및 발판 역할 TERC RNA이며 HOTAIR도 PRC2위한 발판와 13 단지 H3K4 demethylase로 검색할 수 있습니다.

염색질에서 RNA의 인지도를 선행 연구는 상당한 통찰력에게 15,16을 보여주지만, 한 번에 하나의 유전자 현장에있다. 대부분의 lncRNAs의 인 사이트는 알려져 있지되며, 염색질 조절에 lncRNAs의 역할은 주로 lncRNA의 섭동의 간접적인 영향으로부터 유추되었습니다. 염색질의 immunoprecipitation는 microarray 또는 깊은 시퀀싱 (각각 칩 칩 또는 칩 seq) 뒤에 것처럼 크게 게놈 규모의 단백질-DNA 상호 작용에 대한 우리의 이해를 개선하고있다, 우리가 recen을 보여주는tly 고해상도 17 살의 긴 RNA의 인 게놈 전체를 매핑하기위한 전략을 발표했다. 다음과 함께 수백 개의 기지의 해상도 게놈 바인딩 사이트의지도를 생성하는 안티 센스-oligos을 기와로 염색질 복합 : RNA의 정제 (처프) (그림 1)에 의해이 메소드는, 염색질 격리는 대상 lncRNA의 친화 캡쳐를 기반으로 고감도와 배경 쌉니다. 친화력 - 프로브의 설계는 RNA 시퀀스 주어진 간단하고 RNA의 구조 또는 기능적 도메인에 대한 지식을 필요로하지 않기 때문에 처프 다수 lncRNAs에 적용됩니다.

Protocol

1. 프로브 디자인

디자인 안티 센스 DNA는 짹짹에 의해 RNA 대상의 선별 검색을 위해 프로브를 기와.

  1. 에서 온라인 프로브 디자이너를 사용하여 설계 안티 - 감각의 oligo 프로브 singlemoleculefish.com 18.
  2. 이러한 매개 변수를 사용하여 프로브 RNA의 길이 = 1 프로브 / 100 BP의 수 2) 대상의 GC % = 45; 3) Oligonucleotide 길이 = 20; 4) 간격의 길이 = 60-80을. 너무 오래 경우 디자이너에 대한 세그먼트로 RNA 봐라. 반복이나 다양한 상동 지역이 생략하십시오.
  3. 3 소수 끝에 BiotinTEG과 주문 안티 - 감각의 DNA 프로브.
  4. RNA를 따라 자신의 위치에 따라 라벨 프로브. "심지어는"수영장이 모든 프로브에게 번호 2, 4, 6, 등등을 포함하고 "이상한"수영장 1 번호 프로브, 3, 5를 포함 있도록하는 두 개의 수영장으로 그들을 분리 등 100 μm의 농도에 탐사선의 수영장을 희석하고 -20 ° C.에 저장
  5. 모든 실험 사용하여 수행할 수 있습니다서로에 대한 내부 통제의 역할을 둘 다 풀. 프로브 특정 소음 각 풀에 고유 될하면서 리얼 RNA-의존 신호는 두 개의 수영장 선물이 될 것입니다. 이것은 짹짹-qPCR와 짹짹-seq 모두에 적용됩니다.

2. 수확 셀

짹짹 실험에 사용됩니다 세포를 수집합니다.

  1. 조직 배양 플레이트 또는 confluency에 flasks에서 세포를 성장. 인산염과 헹굼 한번 식염수 (PBS)을 버퍼링하고 trypsinize. 세포를 이동시키다 및 단일 세포 현탁액으로 resuspend 아래로, 미디어> 2X 볼륨과 피펫을 트립신을 끄다합니다. 50 ML 팔콘 튜브로 모든 미디어 및 resuspended 세포를 전송합니다. 20,000,000 세포는 일반적으로 한 짹짹 샘플 충분합니다.
  2. 4 분 동안 800RCF에서 세포를 봐. 필요한 경우 PBS 40 ML에서 대기음 매체와 resuspend 40,000,000 세포는 튜브를 결합. 4 분 동안 800RCF에서 세포를 봐. 가만히 따르다 PBS는 조심스럽게 각도 남은 액체에 기음.
  3. 3. 크로스 링크 셀 및 셀 펠릿를 수집

    Crosslink는 RNA-염색질 상호 작용을 유지하고 세포 펠렛을 준비하는 글루 타 알데히드와 세포를 수집했습니다.

    1. 실온에서 모든 단계를 수행합니다.
    2. 방 온도를 PBS에 1 % 글루 타 알데히드를 준비합니다. 10000000 전지 (0.4 ML 25 % 글루 타 알데히드 주식 + 9.6 ML PBS) 당 10 ML을 준비합니다. 글루 타 알데히드는 신선한 사용해야합니다.
    3. 알약을 꺼내려 팔콘 튜브의 바닥을 누릅니다. Resuspend 세포 펠렛은 1 % 글루 타 알데히드에 청크를 피하기 위해 작은 볼륨으로 시작, 그때 전체 볼륨까지 맨. 믹스하는 반전. 엔드 - 투 - 엔드 흔드는 또는 회전에 실온에서 10 분 대한 Crosslink.
    4. 5 분을위한 상온에서 1.25 M 글리신의 1/10th 볼륨 가교 반응을 끄다.
    5. 5 분 동안 2000RCF에서 봐. 20 ML은 5 분 동안 2000RCF에서 방적 한번 PBS를 차게로 뜨는 및 세차 펠렛을 기음.
    6. W를 기음과 resuspend재를 떨어서, 교차 연결된 펠릿 20,000,000 셀 당 한 ML 냉장 PBS로. 주의 깊게 피펫 팁으로 가능한 한 많은 PBS를 제거 4 C.에서 3 분 동안 2000RCF에 Eppendorf 튜브와 스핀 각 ML을 전송합니다.
    7. -80에서 액체 질소와 저장소에있는 세포 알약을 플래시 동결 ° C 무기한.

    4. 세포 용해

    세포 lysate를 준비하는 가교 세포를 Lyse.

    1. 실온에서 냉동 세포 알약을 녹여. 세포 펠렛을 이동시키다하고 혼합하기 위해 최선을 누릅니다. 4에 3 분 ° C.에 대해 2000RCF에 펠렛을 던가 모든 남아있는 PBS를 제거하는 샤프 10 μl 피펫 팁을 사용합니다.
    2. 전자 잔액 (1mg까지 정확하게) 비어있는 Eppendorf 튜브의 살갈퀴 질량에서 (우리의 튜브는 매우 일관되게 1.060 그램 무게). 각 펠렛를 저울질하고 체중을 기록합니다. 가교 HELA 세포의 전체 15cm 요리는 일반적으로 100 MG 무겁죠.
    3. fres와 보충의 용해 완충액 (펠렛의 10X 질량, 100mg에 대한 예 : 1 ML)H 단백 분해 효소 억제제, PMSF와 Superase 인은 (첨부된 버퍼 목록을 참조). 잘 섞는다.
    4. 각각의 튜브로 10X 볼륨 보충 용해 완충액을 넣고 펠렛을 resuspend. 작은 알약 들어 <250 μl 보충 용해 완충액에 resuspend 25 밀리그램. 서스펜션은 부드럽게해야합니다. 그렇지 않다면, 500 μl aliquots으로 정학을 분할하고 대단히 짧은 시간을 깨고 동력 펠렛 믹서를 사용합니다. sonication 즉시 진행합니다.

    5. Sonication

    가교 세포 lysates를 sonicating에 의한 전단의 DNA.

    1. 15 ML 팔콘 튜브에 Bioruptor의 세포 lysate를 Sonicate. 각 튜브에 <1.5 ML의 lysate를 사용하여 빠른 sonication을 위해, 시간에 더 이상 둘을 제외 튜브 sonicate.
    2. 펄스 간격에서 45 초 30 초 최고 설정에서 4 ° C의 물을 욕조에 Sonicate. 매 30 분 lysate 확인합니다. 세포 lysate가 더 이상 흐린되지 않습니다 때까지 sonicating 계속합니다. 이것은 빠르면 30 분, 많은 4와 같은 시간이 걸릴 수 있습니다. 번호튜브, 샘플 볼륨, 목욕 온도 및 sonication 시간의 기간은 프로세스가 걸리는 시간에 영향을줍니다. 튜브 가능성이 서로 다른 속도로 sonicate 때문에 함께 균등을 보장하기 위해 원래의 튜브에 매 30 분 및 재배포 그들을 풀 것이다. 참고 : 글루 타 알데히드 - 가교 세포가 포름 알데히드 등가물보다 sonicate 데 시간이 더 크게 가져가라.
    3. lysate 분명 켜면, 새로운 Eppendorf 튜브에 5 μL lysate를 전송합니다. 90 μL의 DNA 프로 테아제 K (PK) 버퍼 (버퍼리스트 참조) 5 μL PK를 추가합니다. 와동은 믹스 앤 간단히 다운 스핀합니다. 50 45 분 동안 품어 ° C.
    4. Qiagen PCR 정화 키트에 DNA를 추출합니다. Elute의 30 μL Qiagen의 용출 버퍼 (EB)의 DNA와 1 % 아가로 오스 겔에 DNA의 크기를 확인합니다. DNA의 비방의 일괄 100-500 BP 경우, sonication이 완료됩니다. 그렇지 않을 경우 sonicate로 진행합니다.
    5. 4에서 10 분 동안 16100RCF에서 sonicated 샘플을 원심 ° C. 액체 nitroge에 1 ML 샘플 및 플래시 동결로 supernatants, 나누어지는을 결합N. -80시 스토어 ° C.

    6. 짹짹

    바운드 염색질을 RNA와 분리 biotinylated DNA 프로브를 잡종.

    1. 상온에서 염색질의 해동 튜브.
    2. (염색질의 ML 당 2 ML 준비, 버퍼리스트 참조) 하이브리드화 버퍼를 준비합니다. 혼합할 수있는 와동.
    3. lysate 1 ML을 사용하여 일반적인 짹짹 샘플의 경우 Eppendorf 튜브에있는 DNA의 입력과 장소에 대한 RNA의 입력과 10 μL 10 μL를 제거합니다. 추가 사용까지 얼음에 보관.
    4. 15 ML 팔콘 튜브 1 ML의 염색질을 전송합니다. 각 튜브에 두 ML의 하이브리드화 버퍼를 추가합니다. 총 볼륨의 <1.5 ML는 Eppendorf 튜브를 사용합니다.
    5. 실온에서 프로브를 녹여. 당신은 오랫동안 그것을 사용하지 않은 경우에는 금액을 확인 Nanodrop 프로브 (100 μm의 프로브해야 사양 ~ 단일 스트랜드의 DNA 설정을 사용 500-600 NG / μl). 특정 튜브 (1 ML 염색질 당 100 pmol 프로브, 1 ML 염색질 당 100 μL / pmol 프로브 1 μL)에 탐사선의 적절한 볼륨을 추가합니다.잘 섞는다. 떨고있는 4 시간 동안 37 ° C에서 품어.
    6. 20 분이 하이브 리다이 제이션을 위해 남아있는 상태에서 C-1 자석 구슬 (4 ° C에 저장) 준비합니다. 프로브의 100 pmol 당 100 μL를 사용하십시오. 한 ML unsupplemented 용해 버퍼와 버퍼에서 별도의 구슬로 DynaMag-2 자석 스트립을 사용하여 세 번 .. 내사랑.
    7. 용해 완충액의 원래 볼륨에서 Resuspend 구슬, 신선한 PMSF, PI 및 Superase 인과 보충. 4 시간의 하이브 리다이 제이션 반응이 완료되면 각각의 튜브에 100 μL 구슬을 추가합니다. 잘 섞는다. 떨고있는 30 분 동안 37 ° C에서 품어.
    8. (샘플 당 5 ML) 버퍼 씻어 준비합니다. 혼합할 수있는 와동. 37까지 사전 따뜻한 ° C. 사용하기 전에 PMSF를 추가합니다.
    9. 한 ML 세척 버퍼 다섯 번으로 구슬을 씻는다. 첫 번째 세척에서 1, ML 세척 버퍼에 별도의 구슬, 가만히 따르다, 그리고 resuspend에 DynaMag-15 마그네틱 스트립을 사용합니다. 1.5 ML Eppendorf 튜브에 볼륨을 전송합니다. 5 분 동안 흔들어으로 37 ° C에서 품어.
    10. 이후 세차장에서 minicentrifuge 각 튜브를 돌려서, 1 분 DynaMag-2 마그네틱 스트립에 샘플을 설정합니다. 가만히 따르다 예제 1 ML 세척 버퍼에 resuspend Kimwipe로 모든 drips 닦아냅니다. 5 분 동안 흔들어으로 37 ° C에서 품어. 다섯 총 세차장에 대해 반복합니다.
    11. 마지막으로 세척 장에서 잘 구슬 resuspend. 100 μL를 제거하고 RNA 격리를 위해 별도로 설정합니다. DNA 분획을위한 준비 900 μL. DynaMag-2 마그네틱 스트립에있는 모든 튜브를 삽입하고 세차 버퍼를 제거합니다. 간단히 아래 모든 튜브 던가, 자석 스트립에 넣어. 날카로운 10 μl 피펫 팁으로 완전히 세척 버퍼의 마지막 비트를 제거합니다.

    7. RNA 격리

    qRT-PCR에 의해 quantitate하는 처프 샘플에서 RNA 분획을 추출합니다.

    1. 100 μL 비드 샘플과 10 μL RNA 입력 샘플을 가져가라. 입력 RNA 85 μL RNA PK 버퍼 산도 7.0을 추가합니다. 95 μL RNA PK 버퍼 산도 7.0의 Resuspend 구슬. 엔드 - 투 - 엔드 떨고있는 45 분 동안 50 ° C에서 5 μL Proteine​​ase의 K 및 부화를 추가합니다.
    2. 간단히 모든 튜브를 돌려서95에서 열 블록에 10 분 동안 샘플을 끓여서 ° C.
    3. 얼음에 샘플을 풀어, 10 초 동안 적극적으로 500 μL TRIzol, 와동을 추가합니다. 10 분 동안 상온에서 품어. -80시 쇼핑몰 ° C 또는 4 단계로 진행합니다.
    4. TRIzol 처리 샘플에 100 μL의 클로로포름을 추가합니다. 10 초에 대한 노골적인 소용돌이. 4 ° C.에 15 분 benchtop 원심 분리기에 16100RCF에 스핀
    5. 유기 및 인터페이스를 피하 ~ 400 μL 수성 표면에 뜨는을 제거합니다.
    6. 600 μL (1.5 볼륨) 100 % 에탄올을 넣고 잘 섞는다. MIRNeasy 미니 칼럼을 통해 샘플을 봐. RWT (MIRNeasy 미니 키트), 제조 업체의 프로토콜마다 RPE와 2X와 함께 1X 씻으십시오. 30 μL nuclease없는 H 2 O (nfH 2 O)로 Elute.
    7. RNA의 eluate 제조 업체의 프로토콜마다 DNA를 무료로 드셔보세요. 반응이 완료되면, 65에서 15 분 동안 시료를 가열 ° C가 완전히 남아있는 DNase를 inactivate 수 있습니다.
    8. 1 μL RNA를 사용 lncRNA을 확인 qRT-PCR 분석에 당 잘 격리검색. GAPDH는 종종 부정적인 컨트롤로 사용됩니다.

    8. DNA의 격리

    시퀀싱에 의해 식별하거나 qPCR에 의해 quantitate하는 처프 샘플에서 DNA 분획을 추출합니다.

    1. DNA를 용출 버퍼 (버퍼리스트 참조), DNA의 입력을 포함한 샘플 당 150 μl를 준비합니다.
    2. 섞어 한 0μL RNase (10 밀리그램 / ML)와 10 μL RNase H (10 U / μl)의 DNA 용리 버퍼의 ML 당, 그리고 소용돌이를 추가합니다.
    3. RNases있는 DNA를 용출 버퍼 150 μL의 구슬 각각의 샘플을 Resuspend. (140 μL의 Resuspend의 DNA 입력) 떨고있는 30 분 동안 37 ° C에서 품어.
    4. 별도의 구슬과 DynaMag-2 마그네틱 스트립에 뜨는. 뜨는을 제거하고 레이블 튜브에 추가합니다.
    5. 10 μL RNase (10 밀리그램 / ML)와 RNaseH (10 U / μL) 그대로 8.2에서 수행)와 함께 DNA의 용출 버퍼의 두 번째 나누어지는을 준비합니다. 각 샘플 (DNA 입력 포함), 알을 품다 150 μL를 추가하고 뜨는 제거합니다. 모든 뜨는 (shoul를 수집D) ~ 300 μL 수 있습니다.
    6. 각 시료 15 μL PK를 추가합니다. 떨고있는 45 분 동안 50 ° C에서 품어.
    7. 노란색 위상 잠금 젤 튜브 (5PRIME)를 다운 사전 돌리다. 위상 잠금 젤 튜브에의 DNA 샘플을 전송, 300 μL PhOH 추가 샘플 당 Isoamyl : 클로로포름합니다. 10 분 동안 적극적으로 흔들, 4 ° C.에 5 분 대한 16100RCF에서 benchtop 원심 분리기에 다운 스핀 상단 (~ 300 μL)에서 수성을 가져가라. 3 μL GlycoBlue, 30 μL NaOAc, 900 μL 100 % EtOH를 추가합니다. 하룻밤 -20 ° C에서 잘하고 매장 섞는다.
    8. 4에서 30 분 동안 16100RCF에서 샘플을 던가 ° C.
    9. 신중하게 가만히 따르다 뜨는. 한 ML 70 % EtOH와 섞어 소용돌이를 추가합니다. 5 분 동안 16100RCF에서 봐. 피펫으로 뜨는을 제거합니다. 공기 1min 동안 건조. 30 μL EB에 Resuspend.
    10. DNA 샘플은 qPCR 또는 Illumina 프로토콜 당 높은 처리량 시퀀싱 라이브러리의 준비에 의한 분석을 위해 준비가되어 있습니다.

    10. 대표 결과

    그림 1 그림 2 GAPDH 이상 HELA 세포로부터 인간 telomerase RNA (TERC), 부정적인 제어 역할을 풍부한 세포 RNA의 농축을 보여줍니다. GAPDH RNA의 단지 0.46 %가 검색되었습니다 반면 세포에 존재 TERC RNAs (~ 88%)의 대부분은 ~ 200 배로 농축 계수를 보여주 처프를 수행 타고 내려오게되었다. 같은 포유 동물 세포 (그림 2)로 표현되지 않습니다 LacZ RNA를 타겟팅 프로브와 같은 특이 현상이 프로브가 추가로 부정적인 컨트롤로 사용할 수 있습니다.

    대상 lncRNA를 바인딩 것으로 예상의 DNA 영역은 일반적으로 qPCR로 측정 부정적인 영역 통해 풍부합니다. 그림 3은 우리가 같은 세포 라인에서 짹짹-seq를 수행하여 결정하는 주요 인간 포피의 섬유아 세포에서 네번 HOTAIR 바인딩된 사이트 qPCR 검증을 보여줍니다, 반면 TERC와 GAPDH 유전자 사이트 SE부정적인 통제 지역으로 rve. 모두 "도"와 "이상한"탐사선은 부정적인 지역, 진정한 lncRNA 바인딩 사이트의 특징 이상 예상 HOTAIR 바인딩된 사이트 굴복 비교 농축을 설정합니다.

    짹짹 강화 DNA의 높은 처리량 시퀀싱은 lncRNA 바인딩 사이트의 글로벌지도를 얻을. Drosophila lncRNA roX2은 복용량 보상에 필요한 방식으로 X-염색체와 상호 작용하는 것으로 알려져 있습니다. 그림 4는 X의 염색체의 일부분 이상 roX2 바인딩 프로필을 보여줍니다. 모두 "도"와 "이상"샘플은 순서가되어 그들의 독특한 소리가 중복되는 신호의 추적을 생산하기 위해 제거되었습니다. 각각의 "피크"는 여기 roX2 바인딩의 강력한 사이트를 나타냅니다. 전체 트랙과 roX2 대상 유전자의 목록은 추 외에 설명되어있다. 2,011 17가.

    1 그림.
    짹짹 proce의 그림 1. 흐름 차트dure. Biotinylated 기와 프로브는 lncRNA을 대상으로 hybridized되는 생체내에 단백질 부가물을하고, 염색질의 단지는 엄격한 세차장 뒤에는, 자기 streptavidin 비즈를 사용하여 정화됩니다. 염색질은 lncRNA에 가교됩니다. 우리는 putative lncRNA 바인딩 순서가 오렌지색으로 schematized되고 Rnase와 헤의 칵테일과 함께 lncRNA 바운드의 DNA 또는 단백질을 elute. 이전 추 년에 출판된. 2,011. 17

    그림 2.
    인간 TERC RNA 그림 2. 짹짹의 enriches. TERC-asDNA 프로브 세포 TERC RNA 및 탐지 GAPDH의 ~ 88 % 검색할 수 있습니다. LacZ-asDNA 프로브는 부정적인 컨트롤로 사용도 RNAs를 검색할 수 있습니다. 민 + SD가 표시됩니다. 이전 추 년에 출판된. 2,011. 17

    그림 3.
    그림 3. 기본 인간의 HOTAIR 짹짹-qPCR을위한eskin의 섬유아 세포. NFKBIA, HOXD3-4, SERINC5 및 ABCA2가 HOTAIR와 상호 작용하는 지역입니다. TERC와 GAPDH는 부정적인 컨트롤을 역임했습니다. 민 + SD가 표시됩니다. 이전 추 년에 출판된. 2,011. 17

    4 그림.
    그림 4. Sl2 Drosophila 세포의 roX2 RNA의 짹짹-seq 데이터입니다. "비록"과 "이상한"가 별도로 순서가 있었던 것은 그들의 데이터는 모두 전용 일반 봉우리를 반영하도록 병합합니다. 병합된 트랙이 나타납니다. 이전 추 년에 출판된. 2,011. 17

Discussion

여기 짹짹-seq, 생체내 lncRNA 구속력이 사이트는 게놈 전체에 매핑하는 방법을 설명했다. 성공을위한 주요 파라미터는 oligonucleotide 프로브 및 글루 타 알데히드 crosslinking을 기와의 분할 수영장입니다. 친화력 - 프로브의 설계는 RNA 시퀀스 주어진 간단하고 RNA의 구조 또는 기능적 도메인에 대한 사전 지식이 필요하지 않습니다. 두 종족에 세보다는 다른 RNAs - - roX2, TERC 및 HOTAIR와의 성공 짹짹-seq 많은 lncRNAs 대한 가능성 generalizable 것을 제안합니다. 모든 실험과 마찬가지로, 관리 및 적절한 컨트롤이 결과를 해석해야합니다. 다른 lncRNA는 조건 적정과 같은 서로 다른 선호도 프로브 또는 crosslinkers의 선택과 같은 조건의 의식 변화를 요구할 수 있습니다, RNA-염색질 상호 작용의 다양한 측면을 강조할 수 있습니다. 칩 seq 마찬가지로, 모든 바인딩 이벤트가 반드시 기능적 있으며, 추가적인 연구는 RNA O의 생물 학적 결과를 확인하는 데 필요한염색질에 ccupancy. 그럼에도 불구하고, 우리는 수천 8,9의 현재 번호를 다른 염색질 - 관련 lncRNAs의 연구자들은이 기술의 많은 흥미로운 애플 리케이션을 예견한다. 칩 seq는 DNA-단백질 상호 작용의 게놈 차원의 탐험을 위해 문을 열었습니다 것처럼, "RNA interactome"의 짹짹-seq 연구는 생물학의 많은 새로운 길을 보여줄 수 있습니다.

Disclosures

C. 추와 HY 장은이 방법을 바탕으로 특허 출원의 발명가로 지정됩니다.

Acknowledgments

우리는 T. 숙취, MC 감사합니다. 영, O. 매너 E. Segal, M. 구로다, T. Swigut 및 토론을위한 나 Shestopalov. 과학, 기술, 싱가포르 (CC), NIH R01-CA118750 및 R01-HG004361 (HYC) 및 재생 의료 (HYC)의 캘리포니아 공과 대학 연​​구 기관 지원. HYC는 하워드 휴즈 의학 연구소의 조기 채용 과학자이다.

Materials

Name Company Catalog Number Comments
Buffer List:
Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20 °C.
Lysis Buffer:
50 mM Tris-Cl pH 7.0
10 mM EDTA
1% SDS
Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
Proteinase K Buffer (for DNA)
100 mM NaCl
10 mM TrisCl pH 8.0 (For RNA use pH 7.0)
1 mM EDTA
0.5% SDS
Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
Hybridization Buffer
750 mM NaCl
1% SDS
50 mM Tris-Cl pH 7.0
1 mM EDTA
15% formamide (store in the dark at 4 °C)
Always add PMSF, P.I. and Superase-in fresh before use
Wash Buffer
2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock)
0.5% SDS
Always add PMSF fresh before use
DNA elution Buffer
50 mM NaHCO3
1% SDS
Table of specific reagents and equipment:
Glutaraldehyde (EM grade) Sigma-Aldrich G5882-10x10ml
Motorized pellet mixer VWR international V8185-904
Protease inhibitor Roche Group 11873580001
PMSF Sigma-Aldrich 78830
Superase-in Ambion AM2696
Bioruptor Diagenode UCD-200
Falcon tubes (for sonication) Corning 430790
Proteinase K Ambion AM2546
PCR purification kit Qiagen 28106
C-1 magnetic beads Invitrogen 65002
PMSF Sigma-Aldrich P7626-25G
DynaMag-15 magnet Invitrogen 123-01D
DynaMag-2 magnet Invitrogen 123-21D
MIRNeasy mini kit Qiagen 217004
Rnase H Epicentre Biotechnologies R0601K
Rnase A Sigma-Aldrich R4875-100MG
Phase Lock Gel Heavy 5 PRIME 2302810
Trizol Invitrogen 15596-018
Phenol:chloroform:Isoamyl Invitrogen 15593-031
Chloroform Ricca RSOC0020-1C
GlycoBlue Ambion AM9515
Glycine JT Baker 4057-06
PBS, pH 7.4 Invitrogen 10010-049
Elution Buffer (EB) Qiagen 19086
20x SSC Invitrogen 15557-036
10% SDS Invitrogen 15553-027
DNA-free Ambion AM1906
Buffer kit Ambion AM9010
Formamide Invitrogen 15515-026

DOWNLOAD MATERIALS LIST

References

  1. Koziol, M. J., Rinn, J. L. RNA traffic control of chromatin complexes. Curr. Opin. Genet. Dev. 20, 142-148 (2010).
  2. Mercer, T. R., Dinger, M. E., Mattick, J. S. Long non-coding RNAs: insights into functions. Nat. Rev. Genet. 10, 155-159 (2009).
  3. Rinn, J. L. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 129, 1311-1323 (2007).
  4. Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science. 322, 750-756 (2008).
  5. Kelley, R. L. Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell. 98, 513-522 (1999).
  6. Pandey, R. R. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol. Cell. 32, 232-246 (2008).
  7. Wang, K. C. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature. 472, 120-124 (2011).
  8. Khalil, A. M. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. U.S.A. 106, 11667-11672 (2009).
  9. Zhao, J. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell. 40, 939-953 (2010).
  10. Meller, V. H., Wu, K. H., Roman, G., Kuroda, M. I., Davis, R. L. roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system. Cell. 88, 445-457 (1997).
  11. Franke, A., Baker, B. S. The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila. Mol. Cell. 4, 117-122 (1999).
  12. Gupta, R. A. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 464, 1071-1076 (2010).
  13. Tsai, M. C. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 329, 689-693 (2010).
  14. Zappulla, D. C., Cech, T. R. RNA as a flexible scaffold for proteins: yeast telomerase and beyond. Cold Spring Harb. Symp. Quant. Biol.. 71, 217-224 (2006).
  15. Nagano, T. The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science. 322, 1717-1720 (2008).
  16. Carter, D., Chakalova, L., Osborne, C. S., Dai, Y. F., Fraser, P. Long-range chromatin regulatory interactions in vivo. Nature. 32, 623-626 (2002).
  17. Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions. Mol. Cell. (2011).
  18. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods. 5, 877-879 (2008).

Comments

84 Comments

  1. hi,i has a great interest on CHIRP and your works are highly appreciated,but i want to know that where can i find the buffer list?thank you

    Reply
    Posted by: tang n.
    May 5, 2012 - 4:16 AM
  2. Hi there, thanks for the interest in our paper. As you pointed out the original article lacked the buffer list, which we have uploaded a while ago. Hope it helps!

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:20 PM
  3. Hello, I'm trying to perform ChIRP experiments and your protocol is very helpful to me.

    However, I have a question about the final concentration of biotinylated probes and the volume of the beads.
    My probes are 100µM so 1µL corresponds to 100pmol. I have 50 probes and my question is : should I use 100µL of magnetic C1 beads per 100pmol of probe, so in my case, 50 probes x 100µL = 5mL of beads ?

    Thanks a lot for the answer ?

    Reply
    Posted by: Sylvain F.
    October 31, 2012 - 4:47 AM
  4. I also have this question!

    Reply
    Posted by: Guihai f.
    November 28, 2012 - 7:51 AM
  5. Hi guys, thanks for the interest in our paper. That's indeed a common question I get, so apologize for not explaining that more clearly in the paper. The 100uM concentration refers to all probes, so if you have ² probes in the pool the individual concentration is 50uM per probe, and if you have 50 probes it's ²uM per probe. It follows that as you scale up number of probes in the pool you do not have to scale up the amount of beads to add. As a caution though, we have noticed that when I use too many probes the concentration of each probe drops too much and it actually adversely affects yield. Try not to use more than 50 probes if possible.

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:23 PM
  6. what is the fucntion of the Buffer kit ? I can not find it in the protocol.

    Thanks for your time!

    Reply
    Posted by: Guihai f.
    November 1, 2012 - 12:50 AM
  7. The buffer kit is simply a convenient collection of buffers provided by Ambion that includes 5M NaCl, 1M Tris, 0.5M EDTA, etc..

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:25 PM
  8. Hi, I refer to your paper in HOTAIR ChIRP. You used 48 oligo probes for this ncRNA. I am trying to do a HOTAIR ChIRP. I am wondering if the number of probes can be smaller to reduce cost without a huge compromise on specificity. Furthermore, I am not doing sequencing but a specific check on a genomic region of interest, so I supposed the background noise may be lower? Thank you!

    Reply
    Posted by: Yi Fang L.
    November 8, 2012 - 5:11 AM
  9. We share your desire to reduce number of probes used. In preliminary testing I've found that you can use as few as 1 probe per ²00 nucleotide of RNA target RNA. But that number has to be doubled considering if you need both "even" and "odd" set, so effectively tiling density is 1 probe / 100nt.

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:28 PM
  10. Hello,
    I enjoyed your paper very much.

    At the end of the video, and in the figure, you mention that in this process you can isolate RNA binding proteins that were associated with the RNA. My questions are: have you had success in doing this? What is the protocol for isolating the proteins? Do you just isolate the protein fraction from the Trizol, or is there a more specialized method? Is there a paper that may lay out use of this method?

    Thank you for your help.

    Reply
    Posted by: Gavin J.
    January 18, 2013 - 12:09 PM
  11. Hi,
    Great protocol. Just one question. You mention that all ChIRP steps have to be performed at 37C. I can imagine this is important for the hybridisation steps, but I was wondering why this was needed for the washing steps as well?

    Many thanks

    Reply
    Posted by: Sebastian V.
    March 15, 2013 - 10:21 AM
  12. The washing should be performed at 37C just like you would wash a northern blot at elevated temperatures. If this is logistically difficult for you, just warming up the buffer to 37C and washing on a room temp. shaker works well too.

    Reply
    Posted by: Ci C.
    March 15, 2013 - 1:47 PM
  13. Hello,
    Thanks for the detailed and very helpful protocol. Can you tell me what kind of DNA yields to expect from the pull down per input cell amount? I know it will depend on the lncRNA targeted, but I need an idea of what kind of amounts to expect. Could you tell me what you got for your HOTAIR hybridizations? My own hybs only result in a few nanograms of DNA, and I don't know how to determine whether that is nonspecific DNA binding to my beads or whether it is actual lncRNA-bound DNA? How do you control for this? Thank you for your help in advance.

    Reply
    Posted by: blake e.
    March 20, 2013 - 5:44 AM
  14. Hi,

    Congrats for setting up such a protocol. It might have been discussed already but I would like to have an idea about the typical amounts of DNA and RNA that one might expect from 20 x 10 ^6 cells. This of course is transcript specific, but should we expect nanograms, picograms for either DNA and RNA?

    Also have you tried FA instead of glutaraldehyde for x-linking? Many thanks for your time

    Reply
    Posted by: Antonis A.
    November 26, 2013 - 12:15 PM
  15. Hi,

    Thanks for the detailed protocol. I am about to embark on it over the next week or so and had a question regarding the sonication. You advise a Bioruptor for this step however we only have a Covaris available. Would that provide acceptable sonication and do you have any advice regarding appropriate settings?

    Thank you

    Joshua

    Reply
    Posted by: joshua b.
    March 2, 2014 - 12:24 AM
  16. Yes Covaris works, we've found that it generally reduces sonication time by 30-50% compared with Diagenode. Actual time and intensity needs to be optimized for each machine and cell line.

    Reply
    Posted by: Ci C.
    March 3, 2014 - 5:51 PM
  17. Hi,

    Thanks for the detailed protocol. I am about to embark on it over the next week or so and had a question regarding the sonication. You advise a Bioruptor for this step however we only have a Covaris available. Would that provide acceptable sonication and do you have any advice regarding appropriate settings?

    Thank you

    Joshua

    Reply
    Posted by: joshua b.
    March 2, 2014 - 6:32 AM
  18. To all interested ChIRPers, hybridization can be performed overnight instead of 4hours. Doing so greatly reduces hands-on time. Happy ChIRPing!

    Reply
    Posted by: Ci C.
    March 3, 2014 - 8:14 PM
  19. Hi Ci,

    I have one more issue to resolve regarding ChIRPseq that I would appreciate your input on please (or other ChIRPers that have had similar problems).

    We have been unable to produce lysates using glutaraldehyde despite following the protocol to the letter and getting the Sigma brand that is recommended. Even before sonication, the DNA is degraded to a thick band below 200bp. When we try exactly the same process but substitute 1% formaldehyde (which we normally use for ChIP and 3C) or even plain PBS, the DNA is fine. We have even tried doing everything on ice and reducing the centrifuge steps to only 500g but to no avail.

    My questions therefore are:

    1/ Has your team had similar issues with glutaraldehyde in the past or found it to behave quite differently to formaldehyde (apart from needed longer sonication and harder centrifuging)?
    2/ Though you have shown glutaraldehyde to give a better signal to noise ratio, could we still expect to get usable data using 1% formaldehyde or is it not worth the time and effort?

    Thank you for your time

    Joshua Betts

    Reply
    Posted by: joshua b.
    April 16, 2014 - 8:07 AM
  20. Dear Joshua,

    Thanks for your interest in our protocol. It's unfortunate to hear that you're experiencing DNA degradation. We've never experienced this issue, nor have we heard similar problems from our collaborators who successfully did ChIRP. Usually we're more concerned with RNA integrity, naturally, and if you're careful that's not a problem either.

    Could you tell me how you're extracting DNA, especially prior to sonication?

    Regarding alternative crosslinking methods, for certain lncRNAs we've found that strong formaldehyde crosslinking also works (3% for 30min is a good starting point). 1% formaldehyde usually does not work.

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 16, 2014 - 1:55 PM
  21. Dear Chu,

    Thank you for your advice - I will try the protocol again but substitute 3% formaldehyde for the glutaraldehyde. Does that require any other adjustments apart from slower centrifuge spins and shorter sonication times?

    In regards to my DNA isolation, I lysed the cells as per the protocol and then treated with proteinase K for 45mins (at 50deg or 65deg - either seemed ok). I then RNAse treated the sample and either phenol chloroform extracted the DNA or instead used the Qiagen PCR columns before running the product on a 1% agarose gel. The columns gave more of a smear on the gel, whilst the phenol chloroform extraction just had a broad band below 100bp.

    When I performed exactly the same procedures on formaldehyde treated cells, I obtained a bright band of gDNA at the top of the gel as well as a more faint diffuse band below 100bp. This is what we normally see when working with formaldehyde. Am I right to expect a strong band of genomic DNA on the gel when using glutaraldehyde before sonication, or does the glutaraldehyde break the gDNA down during the fixation process? Does your glutaraldehyde fixed sample normally start as a broad smear even prior to sonication?

    Our group has a lot of experience with ChIPseq, 3C and 4Cseq library preparation but we have never dealt with glutaraldehyde before so are unsure what to expect.

    Any further suggestions would be greatly appreciated.

    Thanks

    Joshua

    Reply
    Posted by: joshua b.
    April 24, 2014 - 9:26 AM
  22. Before preparing your ChIRP library for sequencing, do you check your DNA size distribution (for example using Agilent Bioanalyzer). If so, do you see a similar distribution to that seen for a typical ChIP sample (200-800 bp range)?

    Reply
    Posted by: Eric S.
    August 29, 2014 - 2:43 PM
  23. Dear Chu
    I have two questions:
    1.In designing your chirp oligos have you tried to reduce the spacer between biotin-oligonucleotides. Is there any steric reason for choosing 1 probe / 100nt?
    2. Did you tried to crosslink adherent cells directly on plate? we have problem in properly resuspend our differentiated murine myoblasts in PBS+glutaraldehyde.

    thanks in advance
    regards

    Mariangela

    Reply
    Posted by: Mariangela M.
    October 16, 2014 - 9:56 AM
  24. Dear Mariangela,

    1. It's both a cost consideration (fewer probes are cheaper) and also a biochemical noise consideration (more probes introduce higher chances for probe:DNA direct hybridization).

    2. Yes we've found no difference fixing on plate vs. in suspension. Feel free to pick and choose what works best for you.

    Best,
    Chu

    Reply
    Posted by: Ci C.
    October 17, 2014 - 10:43 PM
  25. Dear ChIRP community,

    We are having problems getting ChiRP peaks with our transcripts. We have tried ChiRPing against 4 different transcripts and for all of them we were able to get very good fold enrichments in the genomic locus of each transcript (on average 50 to 200 x over background) capturing native transcription but we cannot find other binding sites in the genome for any of them. We have good reasons to believe that some or all of our transcripts are chromatin localized and we also know that the signal in the genomic loci is RNA dependent because it is dramatically reduced when we ChiRP after knocking down our transcripts of interest.

    Our transcripts are low in expression, on average 3 to 10 copies per cell. Any ideas as to how can we improve/solve the issue? Is it abundance that is limiting the system? Has anyone experienced/solved something similar?
    We tried the normal protocol, and now we are trying with 3 % FA in parallel to glut. Any suggestions would be very welcomed!

    Antonis

    Reply
    Posted by: Antonis A.
    November 13, 2014 - 5:18 AM
  26. Hi Antonis, I'm afraid the copy number is simply too low. In all of the ChIRP paper published so far I don't think anyone went that low.

    Your enrichment of the genomic loci could be complicated by ChIRP probes pulling down the DNA directly. Do an RNase-control to see if the signal is still there.

    Reply
    Posted by: Ci C.
    November 13, 2014 - 8:52 AM
  27. Dear Chu,

    Thank you for the prompt reply-yes I know that with such expression levels, ChIRP is challenging. I can try the RNase control but as mentioned I am confident about the RNA-dependence of the signal, because it falls back to LacZ levels when I knock down the transcripts. I guess we will have to try increasing the sensitivity of the experiment. Could an increase in x-linking time help here, have you guys tried 20 min of glut x-linking?

    Reply
    Posted by: Antonis A.
    November 18, 2014 - 3:28 AM
  28. Hello,
    I enjoyed your paper very much.

    At the end of the video, and in the figure, you mention that in this process you can isolate RNA binding proteins that were associated with the RNA. My questions are: have you had success in doing this? What is the protocol for isolating the proteins? Do you just isolate the protein fraction from the Trizol, or is there a more specialized method? Is there a paper that may lay out use of this method?

    Thank you for your help.

    Reply
    Posted by: jason h.
    November 26, 2014 - 10:15 PM
  29. Dear ChIRPers, we're happy to announce that you can now officially design and order ChIRP from Biosearch Technologies (Single molecule fish). Scientists developing ChIRP probes using the Stellaris probe designer and following the guidelines from our publication can now email Sheila Semaan at Biosearch Technologies, Inc. for help with ordering the probes in the proper format. Sheila’s contact info is as follows:

    Sheila J. Semaan, Ph.D., Associate Product Manager
    Biosearch Technologies, Inc.
    Email: ssemaan@biosearchtech.com

    Reply
    Posted by: Ci C.
    December 17, 2014 - 9:33 AM
  30. Dear ChIRP community,

    You can soon study RNA-protein interactions in vivo using ChIRP-mass spec. The story is coming out in Cell on April 2nd. Stay tuned!

    Reply
    Posted by: Ci C.
    March 25, 2015 - 5:42 AM
  31. Hello, thank you for the protocol! I also want to perform a RNA pulldown using your protocol and i am very excited for your RNA-protein interaction protocol! I have one question regarding the biotin oligos. Do they need to be 3' biotinylated or can they also be 5' biotinylated. Have you tested this? And you say nothing about (salt, HPLC) purification of the oligos. Thanks for answering my questions.

    Reply
    Posted by: Christine C.
    March 30, 2015 - 10:01 AM
  32. and is it necessary to order Biotin-TEG oligos or is "standard" biotin sufficient?

    Reply
    Posted by: Christine C.
    March 30, 2015 - 11:33 AM
  33. Hi Christine, thanks for your interest in our technique. Regarding your questions:

    1) 3' biotinylation is preferred, because oligos are synthesized 3' to 5'. This way, we ensure all oligos start with a biotin tag. 5' biotin tags are usually added to a synthesized oligo, and that increases the chance of untagged probes, which is less than ideal. However if you can ensure high tagging efficiency on 5' end, and the cost is advantageous, I don't see why it won't work!

    2) We use a disposable reverse phase column called "Glen-Pak DNA purification cartridge," which is a very efficient and cost-effective system. These are really short DNA probes and HPLC or PAGE would be necessary (also probably can't tell tag from untag probes).

    3) TEG ensures no steric hindrance, I don't have hard evidence on this but I think it's necessary.

    Reply
    Posted by: Ci C.
    March 30, 2015 - 10:03 PM
  34. Thank you for your answers! As I am performing your protocol with the aim of an RNA-protein pulldown, i have some other questions regarding the protocol, if you don't mind.

    - in the protocol before washed your magnetic beads in unsupplemented lysis buffer and you blocked the magnetic beads using BSA and yeast RNA- in the latest protocol you don't block. Why is that?

    - ChIRP-MS requires 100 Mio cells, ChIRP-seq 20 Mio cells. If i want to perform ChIRP-MS (or western blot) do i need 100 pmol of probe for each 20 Mio cells --> 500 pmol of probe for ChIRP-MS? --> 500 µl beads(??)

    Reply
    Posted by: Christine C.
    April 8, 2015 - 8:12 AM
  35. Hi Christine,

    - We no longer block as there's no difference in results, and we prefer a more streamlined protocol. BSA would also potentially contaminate ms results.

    - yes scale up beads accordingly. We do 1ml beads per experiment routinely. It gets expensive, but hopefully you'd only have to do it once or twice, and validate with western in a smaller scale later. For western of more abundant chirp hits, I do from 1/5 of cell pellets, but for less abundant ones I still use full amount.

    Reply
    Posted by: Ci C.
    April 8, 2015 - 11:36 PM
  36. Dear ChIRPers,

    ChIRP mass spec has just come online today at Cell. We're very pleased to introduce a very robust and straight-forward method that's optimized to study lncRNA:protein interactions. Check out the story at:

    http://www.cell.com/cell/abstract/S0092-8674(15)00312-8

    Happy ChIRPing!

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 2, 2015 - 9:00 PM
  37. Dear ChIRPers,

    Now you can easily design and order ChIRP probes with the latest optimization from Biosearch Technologies, the company that invented Single Molecule FISH.

    https://www.biosearchtech.com/chirpdesigner/

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 2, 2015 - 10:24 PM
  38. Hi, i'm trying to perform ChIRP-western and i have some questions concerning the methods.

    1) for a control, i also want to see if my RNA is present: how can i get a small aliquot of post-ChIRP beads to perform RNA elution with this aliquot
    2) for the protein biotin elution buffer: resuspend beads in what volume?
    3) for ChIRP-MS lysates are incubated with 30µl beads - can i reuse them?

    thank you in advance!

    Reply
    Posted by: Carolin C.
    April 14, 2015 - 7:18 AM
  39. Hi Carolin,

    thank you for your interested in our method.

    1) yes that's what we we as well. Simply take a 1% or 10% aliquot of your post-wash beads, and extract RNA.

    2) that's dependent on your starting beads volume. But there's a practical constraint, you don't want to have so much volume that after addition of TCA will exceed the capacity of an eppendorf tube.

    3) I'm not sure if I understand your question. Reuse lysate or beads?

    Reply
    Posted by: Ci C.
    April 15, 2015 - 12:20 AM
  40. thank you for the answers!
    to 2) again: if my beads are 100µl starting volume - so i resuspend 2x in biotin elution buffer and pool. Is 2x100µl then enough? and then add 25% of 100%TCA (50µl)?
    to 3) my question is, if i reuse the beads in the hybridization step. and just add another 70 µl beads (=100µl beads)

    Reply
    Posted by: Carolin C.
    April 24, 2015 - 11:23 AM
  41. so what do you recommend for my previous questions 2) and 3)

    another question: how do you calculate the amount of RNA retrieved? I retrieve only 10% of my RNA of interest calculated as % of Input. Furthermore,when i took a sample of what hasn't bound (after hybridization and capture of the beads) i get only 15% (fraction of washing doesn't include much RNA). so where has my RNA gone? i repeated several times… degradation?

    Reply
    Posted by: Carolin C.
    May 20, 2015 - 5:13 AM
  42. Hi, Carolin
    I am also, using ChiRP. Can you tell me the equation you used to calculate the % of your retrieval

    thanks

    Reply
    Posted by: u.
    June 28, 2016 - 1:22 AM
  43. Hi Carolin, May I ask in the end with your 10-20% retrieval rate of RNA, can you identify new proteins by MS via UV crosslink? Thanks.

    Reply
    Posted by: Zhenqiu H.
    May 17, 2018 - 6:20 PM
  44. Dear ChIRPers, we're glad to introduce the Magna ChIRP kit from Millipore. It's a simple pre-made reagent set for scientists who prefer the reliability and user-friendliness of a reputable brand.

    https://www.emdmillipore.com/US/en/product/EZ--Magna-ChIRP-RNA-Interactome-Kit---Isolation-and-characterization-of-non-coding-RNA%3Achromatin-complexes,MM_NF-17-10495

    Happy ChIRPing!

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 15, 2015 - 12:35 AM
  45. Hi Chu

    Thanks for sharing this information.
    Did anyone try this kit already and how good is it? Kits are easy to use and save time for reagents.

    Reply
    Posted by: Liu Z.
    April 16, 2015 - 5:17 PM
  46. Dear Chu,

    Many thanks for your excellent protocol. I'm designing a probe set and have been looking at the probes you designed for HOTAIR and XIST for inspiration. I noticed that some of the probes designed for XIST target the sequence at multiple loci (e.g. Probe 10), albeit ocassionally with a single base mismatch. Either way, I assume this may reduce the number of probes needed. Could I ask whether this was a deliberate design strategy and, if so, does the Biosearchtech ChIRP designer incorporate this strategy?

    Thanks

    Reply
    Posted by: Sebastian V.
    April 16, 2015 - 11:31 AM
  47. Dear Chu

    I feel exciting when I am reading your chirp paper in molecular cell. I have 2 questions that need your input.

    1) Have you or others you know tried in vivo tissue samples and if so how it works? I realized that your molecular cell and the latest cell paper used cell lines primarily.

    2) If I have a non-coding RNA which is 400 bp alone, and cannot generate more than 8 probes based on the parameters provided by https://www.biosearchtech.com/chirpdesigner/ ? What should I do ? Could I just reduce the spacer length between odd and even probes ? do you think it will increase background due to the increased probe density?

    Thanks

    Zhiyong

    Reply
    Posted by: Liu Z.
    April 16, 2015 - 4:49 PM
  48. Hi Zhiyong,

    Thank you for your kind words about our paper. Regarding your questions:

    1) Yes we have ChIRPed in drosophila tissues with great success. The key is probably to have small enough chunks of tissue that can be crosslinked effectively and thoroughly.

    2) Email the Biosearchtech team and they can help you manually reduce the number of probes.

    Good luck!

    Best,
    Chu

    Reply
    Posted by: Ci C.
    May 22, 2015 - 11:15 PM
  49. hey there, i was able to retrieve RNA with your method, but i'm not retrieving RNA bound protein (even though i did see a milky pellet after TCA and acetone treatment). i cross link with UV but the other steps i perform are just the same you are doing.
    i'm not so sure about this competetive elution method by using free d-biotin - and do you no longer add protease inhibitors in your buffer? the beads are simply eluted by using biotin buffer, rotate sample for 20 min,RT and 10 min mixing at 65°C (x2). This is it? then everything should be in my buffer and i continue with TCA precipitation ON.
    sorry if i this sounds stupid to you. i'm just looking for mistakes i could have made...

    Reply
    Posted by: Carolin C.
    June 10, 2015 - 10:32 AM
  50. Hi Carolin, I'm glad you're able to get RNA yield, that's a very good sign. The reason that you're not getting proteins could be: 1) mass spec instrument not sensitive enough. The quality of your results varies A LOT with who runs your mass spec. We've had amazing experience with Bill Lane at Harvard. 2) UV cross linking is known to be super low yield. Unless your lncRNA is very abundant or you're prepared to start with a huge amount of material, I would suggest formaldehyde as your first step. 3) TCA and cold acetone with give you a tiny pellet no matter what (the detergent acts as a carrier that will precipitate by itself even in the absence of proteins). So unless you see a sizable pellet, the presence of a pellet itself doesn't mean anything. d-biotin elution is complete, assuming you're using the C1 beads and biotin from invitrogen. Protease inhibitors are absent at the elution step, because your solution should be quite pure and free of contaminating proteases at that point assuming good lab practice. Hope this helps!

    Reply
    Posted by: Ci C.
    June 10, 2015 - 5:45 PM
  51. hey ci! thank you for your answers!
    1) i'm doing western blot as a start, later on i will do mass spec - so thank you for your tip&hint! as i already know a 100% candidate to get retrieved with my (cytoplasmic) mRNA, i stained for this candidate and got no result.
    2) you also mentioned uv-crosslinking in your first protocol, do you have a comment or any experience on the time of cross linking or amount of joules? so far i cross linked 150mj/cm^2 but I'm thinking to increase that. also i need to scale up my cells/beads/probe as you're saying. i do not know if formaldehyde cross linking works in my case, as i have cytosolic mrna.
    3) yes I'm using c1 dynabeads and d-biotin from invitrogen! thanks for the explanation, that helps!

    thank you very much for your quick answers, tips and hints!

    Reply
    Posted by: Carolin C.
    June 11, 2015 - 8:43 AM
  52. hey there, repeated the experiment - this time i used 200 Mio MEF cells - 10 ml lysate and 1 ml beads, 10µl probe. i also increased cross linking to 300 mj/cm^2 - still no proteins (known RNA binding protein) detectable via western blot. i confirmed RNA pulldown via qPCR after cross linking from a small fraction of beads.
    is sonication necessary?
    any troubleshooting ideas?

    Reply
    Posted by: Carolin C.
    June 17, 2015 - 11:38 AM
  53. I would start with formaldehyde first. UV is known to cross link very inefficiently, and certain residues of contact may not be amenable to photocrosslinking at all. Yes sonication is necessary to soluble the cell lysate.

    Reply
    Posted by: Ci C.
    June 19, 2015 - 2:37 PM
  54. i'm using your 25%TCA / Aceton precipitation method to precipitate my proteins. Have you also experienced a pH change (when adding laemmli it turns green/yellow) due to rest TCA? And i think i don't get my pellet resuspended - even after mixing and resuspending. Isn't boiling for 30 min, 95°C destroying the proteins?

    Posted by: Carolin C.
    July 10, 2015 - 7:30 AM
  55. Yes I have had similar experiences before: pH change usually can be prevented by more careful and thorough acetone washes (although need to care not to disturb pellet), and over drying will cause difficulty in resuspension. I never air dry for over a minute. Boiling is completely fine. Try using the LDS samples buffer / Bis-tris gel system from invitrogen, these work great on chirp samples. If you are still having issues with pH, you can manually adjust a little with a moderate alkaline buffer.

    Posted by: Ci C.
    July 10, 2015 - 10:59 PM
  56. Hi Carolin,

    I've the same problem as you had. I could only retrieve 10-15% of RNA when compare to input RNA. Could you please share how you sort this problem?

    Thank you in advance!

    Reply
    Posted by: Ganeshkumar A.
    July 23, 2015 - 4:25 AM
  57. Hi Ganeshkumar, unfortunately i didn't solve the problem… but i found more RNA of interest in my beads when i directly resuspended them in trizol (leaving out proteinase K treatment) - since i'm interested in my proteins bound to the RNA the proteinase K treatment step is of no importance to me. But so far i still couldn't retrieve my RNA bound proteins.

    Reply
    Posted by: Carolin C.
    July 23, 2015 - 5:19 AM
  58. Hi Carolin,

    Thanks for your immediate reply. I'll also avoid Proteinase K step and try again.
    I've one more question. We always do two step RT-qPCR. So, the amount of RNA that I retrieve isn't enough to perform a RT reaction. How do you do your RT-qPCR? This question maybe a bit stupid, but i really wanna sort out all the issues as early as possible.

    Thanks in advance.

    Reply
    Posted by: Ganeshkumar A.
    July 23, 2015 - 5:27 AM
  59. Hello,
    my RNA amount is also quite low, measured by Nanodrop (Once i measured with qubit and could't get anything). I'm using max. volume possible for RT reaction and adapt for input, not bound… Then i perform qPCR with my cDNA

    But i suppose you cannot skip proteinase K step when your lysate is formaldehyde treated, or you need to boil it first to reverse the crosslink. i diluted my beads in trizol when i had non-crosslinked samples to check for retrieval percentage

    Posted by: Carolin C.
    July 23, 2015 - 7:12 AM
  60. We always do a one-step qRT-PCR (i.e. each qPCR well runs it's own RT). This in my hands is much more sensitive and convenient as well (you don't have to measure RNA or cDNA, just load equal portions of each sample). We use Strategene Brilliant II SYBR reagents and Roche 480 machine. Let me know if this helps with yield detected.

    Posted by: Ci C.
    August 4, 2015 - 8:32 AM
  61. Hi Ci,

    I did gene specific cDNA synthesis with 10ng of RNA from all conditions and treated with RNase H and A (in excess) and then proceeded to qPCR. This worked good for me and I'm getting around 14-25 fold enrichment in my odd and even probe samples when compared to input.

    Thank you and Carolin for your suggestions.

    Posted by: Ganeshkumar A.
    August 4, 2015 - 10:43 AM
  62. Hello,
    i have not yet retrieved any RNA bound protein, so i'm thinking about what issues to sort out. i have to say i can retrieve my RNA of interest (but only max. 20% of input)

    i'm wondering about the protein amount in your formaldehyde cross linked, sonicated and hypotone lysed cell lysats. I experienced the protein amount (measured with Bradford) to be 5-8 fold lower compared to uncross linked or Uv cross linked cells treated with a NP40 lysis buffer.
    Why is that, and will it affect my experiments? Any ideas why the protein amount is so low?
    Thank you in advance!

    Reply
    Posted by: Christine C.
    July 23, 2015 - 5:47 AM
  63. Hi Christine, off the top of my head the two main reason for less protein detected upon cross linking is 1) insufficient sonication, and 2) insufficient reverse cross linking (i.e. boiling). Regarding 1), you have to titrate sonication time of your lysate, and pick the minimum sonication required to release all proteins and RNA of interest. To test this, precipitate the lysate post sonication for 10 min at max speed, and run qRTPCR against your RNA of interest and western against the protein on both the supernatant and pellet, the latter resuspended in appropriate buffer. This way you'll know if there's anything left to be solubilized. 3% cross linked lysates can take significantly longer to solublized than your typical ChIP cross linking.

    Regarding 2), do no less than 30min boiling in sample buffer. I've titrate that also, and 10min or 20min recovers much less protein.

    Let me know if these help and let's start from here. Good luck!

    Reply
    Posted by: Ci C.
    August 4, 2015 - 8:29 AM
  64. Hello,
    Many thanks again for this superb protocol. I'm currently trying to optimise this for mRNA pull-down from fixed cells. I've tried various fixing conditions in parallel and dot-blotted for my RBP of interest vs a non-binding protein (GAPDH) after performing ChIRP. I found that 1% glutaraldehyde (10 min) samples blotted strongly for my RBP, but produced an equally strong signal for GAPDH. The latter was also the case for 3% formaldehyde (10 min), but blots of both GAPDH and my RBP weren't as strong. The only sample that showed a slightly stronger signal of the RBP vs GAPDH was 1% formaldehyde (10 min), but both signals were quite weak. All samples were sonicated for several 30 sec ON and 45 OFF cycles at maximum intensity before foaming to produce sheared RNA of <500 bp in optically clear samples.

    I tested the expression of mRNA of interest in samples pulled down with my tiling oligo set compared with a run using non-targeting scrabled oligos. A capture on unfixed cells produced a very high target yield in the tiling oligo sample and no mRNA in the scrambled control sample. However, performing the same pull down in the 1% formaldehyde-fixed+sonicated samples dramatically increased background (GAPDH mRNA) compared to the scrambled control. I'm curious if you found anything similar during the development of ChIRP and ChIRP-MS and if you might have any solutions? Would extra sonication perhaps help?

    Many thanks

    Sebastian Vencken PhD
    Post-Doctoral Researcher

    Reply
    Posted by: Sebastian V.
    September 13, 2015 - 8:06 PM
  65. Hi Sebastian,

    Have you had any luck adapting this technique to mRNA capture?

    Best,
    Mike

    Reply
    Posted by: Michael K.
    December 7, 2016 - 4:48 PM
  66. Mixed results to be honest. We went forward with 1% formaldehyde and tried different forms of sonication (incl. covaris which didn't give us effective fragmentation). Biased background was still an issue and the copurification of our RBP (Ago+miRNA) was much lower than expected. This has apparently successfully been performed before (https://www.ncbi.nlm.nih.gov/pubmed/23325846) but we didn't manage to reproduce these results for our target.

    Reply
    Posted by: Sebastian V.
    December 8, 2016 - 4:15 AM
  67. Thanks for the feedback. Does your miR-CATCH work well/better? Similar idea, no sonication, and probes attached to beads first. Have you ever tried to use this to look for bound proteins?

    I also ran across this mRNA pulldown from yeast (https://www.ncbi.nlm.nih.gov/pubmed/27641505). They also attached the oligos to the beads first, but the buffers look more like ChIRP.

    Reply
    Posted by: Michael K.
    December 8, 2016 - 11:44 AM
  68. We've had some pretty good results (and publications) from miR-CATCH, but have also discovered that the performance can be very dependent on both the target and oligo design.
    With thorough optimisation we managed to reduce background in miR-CATCH, but each target was a new challenge in this respect.

    We hoped that ChIRP would significantly reduce the issue of background, but we have found balancing fragmentation and signal to be difficult. Dot blots for Ago2 gave us very high background signal in scrambled controls when using gluteraldehyde, while 1% formaldehyde gave some better signal differentiation, but at reduced strength. If I'd take ChIRP further for mRNA:RBP(:miRNA) capture, I'd look at the sonication and fixing steps again as there may be further room for improvement. Unfortunately, I've moved to other things for now.

    Reply
    Posted by: Sebastian V.
    December 9, 2016 - 10:42 AM
  69. Hello!

    Many thanks for the detailed protocol, however i am having trouble at crosslinking and sonication steps! I am sorry if it sounds silly but I just want to identifiy what I am missing/doing wrong.

    In the near future, I want to scale up for ChIRP MS, however first want to get these steps right as CHIRP MS would consume 10 times the beads.

    Crosslinking - When I add Glycine to quench formaldehyde, it doesnt change color?

    Sonication - As used by your team, I am using Bioruptor to sonicate the Hela S3 lysate (20 Mi. cells/1ml supplemeted lysis buffer) in 15 ml falcon (Corning) with a probe attached to the cap. I cant seem to get a clear lysate as shown in your video. I have tried sonicating starting from 30 cycles to 300 cycles and it just froths towards the end. After centrifugation to remove cell debris, the lysate is still considerably cloudy.

    I see that a lot of people are working on optimising this method and would appreciate any tips / tricks that might help me get going.

    Looking forward to some suggestions.

    Best regards,
    Minakshi
    PhD Student

    Reply
    Posted by: Minakshi G.
    September 30, 2015 - 5:06 AM
  70. Hi Minakshi, thanks for your interest in our protocol. Re your questions:

    - glycine doesn't change the color of formaldehyde, just glutaraldehyde.
    - check our bioruptor to make sure power is normal and probes are centered correctly. It shouldn't take more than a couple hours even for hardy cell lines. Frosting is strange and indicates to me that you may not be centering the probe correctly and thus losing efficiency.

    Reply
    Posted by: Ci C.
    November 2, 2015 - 2:46 PM
  71. Hi,
    I would have a question regarding the usage of formamide. My concern is that once the bottle is opened and the
    formamide is exposed to oxygen, it will begin to oxidize to formic acid. Do you take any precautions to prevent oxidation? How do you store formamide for further use? Sigma suggests to purge formamide with nitrogen and store it frozen to prevent oxidation.

    Thanks a lot for your help.

    Best wishes,
    matjaz

    Reply
    Posted by: Matjaz B.
    November 2, 2015 - 4:53 AM
  72. Hi Matjaz, thank you for your interest in our technique. formamide is indeed subject to oxidation and ionization. Re-deionization is laborious, so we tried our best to protect the reagents by parafilming around the bottle cap after each use and store it at 4C. I usually run through a 1 liter bottle in a year with repeated opening and closing, and have not observed any effect on results over time. Hope this helps.

    Reply
    Posted by: Ci C.
    November 2, 2015 - 2:44 PM
  73. Hello,

    I have another technical question. Even though our probes are very specific (we detect target RNA using different probes with high specificity), we have problem with high DNA background signal. Have you also had a similar problem? One way to resolve the issue would be to pre-clear lysates with beads before the hybridisation step. Have you ever implemented this step? If yes, have you done the pre-clearing in lysis buffer with or without hybridisation buffer?

    Many thanks,

    - matjaz

    Reply
    Posted by: Matjaz B.
    November 3, 2015 - 7:36 AM
  74. Hi Ci,

    Thanks for providing such an excellent protocol. I have a question about probe design. The 3' half of lncRNA I interested in (5kb in length) was fully cover by repeat elements. Because ChIRP probe Designer ignored the repeat sequence when designing probe, I found almost no probe target to the 3' half of this lncRNA. I'm afraid that I will miss all proteins bind to the 3' end of lncRNA if I only use the probe target to the 5' end of lncRNA. I wonder how do you handle lncRNA contains large number of repeat sequence?

    Thanks for any help.

    Sincerely,
    Jian-You

    Reply
    Posted by: Jian-You L.
    December 3, 2015 - 9:48 PM
  75. Hi all!

    Thank you for sharing this protocol!
    I have just analysed the RT-qPCR results coming from the RNA samples in order to analyse the lncRNA retrieval and I have retrieved a very small percentage of the lncRNA. From the input and the ChIRP samples I have used the same volume of sample (for the qPCR, around 36ng for the input and 21g for the ChIRP) and I have obtained a Ct of 30 for the input and 37 for the ChIRP measuring my lncRNA of interest. How do you normalise the results? Do you use the same volume or ng of the different RNA samples? Do you use a normalisation method similar to the ChIP one? https://www.thermofisher.com/uk/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html

    Thank you in advance

    Isabel

    Reply
    Posted by: Isabel R.
    December 17, 2015 - 9:30 AM
  76. Hi, Isabel
    we experience along time to optimize the protocol in our lab around 9 month for lncRNA and we found the result unstable for different lncRNAs but for your normalization we usually use a normalized methods like Chip experiment for both GAPDH and our target,
    Hope that helps
    for more contact amr@mail.ustc.edu.cn

    Reply
    Posted by: u.
    December 8, 2016 - 7:17 PM
  77. Dear Chu:
    Recently I'm trying to perform ChIRP experiments and your detailed protocol is very helpful. However, I have some questions about cell lysis and sonication.
    1. I find that the supplemented lysis buffer cannot lyse the crosslinked cells. The crosslinked cells are only resuspended, after a few minutes they will sink to the bottom of the tube, is it normal?
    2. You emphasize that lysis buffer should be added fresh Protease Inhibitor, PMSF and Superase-in then proceed immediately to sonication after resuspended smooth, however, a sample will be added appropriate supplemented lysis buffer in proportion then devided into several parts to sonicate by limiting ultrasound equipment, and will it be affected?
    3. I only have a Covaris available for sonication in lab, I tried many times but could not find an appropriate setting. My DNA size distribution is normally a bright band of gDNA at the top, a bright band above 2000bp and a diffuse band from 2000bp to 100bp, if I continued to sonicate, the diffuse band would below 100bp, and there were considerably cloudy after centrifuged the sonicated samples. I want to know what and how much influence if DNA smear is less than 100bp or more than 500 bp? Do you have any advice regarding it?
    Looking forward to some suggestions, thank you in advance!
    Best regards
    mixue
    Ph.D. Student

    Reply
    Posted by: he j.
    July 17, 2016 - 3:37 AM
  78. Dear Dr CHU.
    First of all, sorry for my english, but I'm french.
    I work on lncRNA and I really appreciate your two articles on ChIRP and Chirp-MS, and since some months I try to set it up, but unfortunately without result until now.
    => I designed the probes as you know but my lncRNA is about 2.3kb so i was able to design only 26 probes. I also ordered your Positive and Negative Control.
    => I amplified the cells until 200 to 300 million and I cross linked them with 3% formaldehyde 30min and stop it with Glycine 0.125M 5 to 10 min, as you mention.
    => My sonication was tested in order to have a smear of DNA between 100 and 600bp and it took between 120 and 140min. I use a Bioruptore in cold room and I have to change water and ice every 15min in order to maintain the low temperature. Samples are centrifuged and debris are removed.
    => I use 2 to 3 aliquots of 1mL of lysed cells for each test, and the indicated quantity of probes. Of course before incubation, samples are pre-incubated with prewashed beads.
    => First incubation is done at 37 ° C in a shaking system, overnight. Second incubation (after addition of the prewashed beads) is done at 37 ° C for 1 hour.
    => Wash is done 5 times with wash buffer, then samples are eluted (using elution buffer freshly prepared with Sigma-Aldrich Biotine) and precipitated with TCA and Aceton. Pellet are directly dissolved in Laemmli, incubated 30min at 95°C and loaded with 12% acrylamide gel. After 1h at 200V the gel is stained with Coomassie Blue and destained 3 times 45min.
    I found NO BANDS even with Positive and negative controls, but after elution, when I checked the presence of my lncRNA in the sample selected with my probes, the result is positive.

    Could you tell me what are the possible mistakes that I could do ....?
    Thanks a lot.

    Reply
    Posted by: Sebastien C.
    January 3, 2017 - 5:02 AM
  79. Dear Dr. Chu,

    Good day! I am Anchilie, a PhD student, and I am really interested in applying your discovered technique in plants. I am currently doing the ChiRP-MS in Arabidopsis but I don't seem to get any RNA-bound proteins in my sample.

    I would just like to briefly tell you how I do the ChiRP-MS in plants. First, I harvest 3g of 14-day old seedlings and crosslinked it with 1% formaldehyde. From these, I then do nuclear extraction and do sonication. From the 1mL sonicated chromatin, I dilute it with 2mL of hybridization buffer, add 1ul probe and 100ul of beads and incubated then in 37C for O/N or 4 hours at 37C. From then I wash the beads 5x and precipitated the proteins using the biotin elution buffer and TCA method. The samples in 1x Laemmli buffer were submitted to LC/MS. I also check the enrichment in the qPCR and are enriched by 500-fold. However, upon analyzing my samples, the mass spec results only detected mitochondrial and chloroplastic proteins in the control and non-coding RNA

    I also tried many things like using 3% formaldehyde and UV crosslinking. I also did direct boiling of beads in Laemmli buffer. I also tried increasing the amount of material 10x accompanied with increasing the probe and the beads.
    But it doesn't really help.

    I would really appreciate to receive some feedback from you and your group with this matter.

    Best regards,
    Anchilie

    Reply
    Posted by: Anchilie M.
    February 27, 2017 - 4:49 AM
  80. Dear Anchilie,

    I am doing CHIRP in arabidopsis, you are the only only doing CHIRP as far as I know. I met some problems. I couldn't enrich my non coding RNA. So I want to know how did you do your CHIRP? Could you please share your protocol with me? Thanks!

    Reply
    Posted by: Zhi-Hong L.
    June 19, 2017 - 7:56 AM
  81. Hi guys, can anyone help me how the percentage calculation for lncRNA retrieval is done.

    Thank you

    Reply
    Posted by: Sunil B.
    June 22, 2017 - 3:58 PM
  82. Hi,

    Thank you for sharing this protocol, I have a question. If I want to study the potential interaction between a protein and a long non coding RNA in rDNA locus (or eventually with the rDNA directly), do you think that your kit will work ?
    Thank you for your answer

    Reply
    Posted by: Burette M.
    April 12, 2018 - 3:27 AM
  83. Dear professor,
    Thank you for sharing this protocol! It helps me a lot. I have a question to ask. Since ultrasound may break the hydrogen bond, I want to ask whether it is necessary to perform sonication when I study weak RNA-RNA interaction, for example: lncRNA-miRNA interaction?

    Reply
    Posted by: Zihao L.
    May 26, 2018 - 10:52 PM
  84. Dear professor,

    Any idea if using a probe-based sonication (like the Branson sonifier) would work? Anyone has tried it ?
    in the previous lab, we were using the bioruptor however we don't have it where I am now unfortunately and needed help to optimize it on the Branson or probe-based sonifiers?

    Thanks

    Reply
    Posted by: Anonymous
    March 1, 2020 - 4:26 AM

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