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lncRNA靶点筛选的利器——CHIRP、CHART、RAP

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发表于 2016-1-29 11:57:56 | 显示全部楼层 |阅读模式

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        lncRNA已经不再是科研领域的新星,芯片、Seq等技术的应用,让“差异lncRNA”筛选之风盛行已久。然而,当获得差异lncRNA以后,如何继续往下游探讨,研究其作用机制呢?生物信息学预测为大家提供了很多的线索和思路,但是除此之外,有没有更好的“一击制敌”,不用预测,用实验数据讲故事的方法?下面的内容,就为大家分享一下,lncRNA靶点筛选的利器。

图片.png

    Schematic representation of ChIRP, CHART and RAP to identify associated proteins and chromatin DNA. RNA–protein–DNA complexes were cross-linked in vivo and solubilized by sonication. Corresponding biotinylated oligonucleotides of three methods were designed and synthesized to be hybridized to target lncRNAs under stringent conditions. Associated proteins and chromatin DNA were efficiently pulled down with streptavidin

magnetic beads. Co-purified RNA, protein and DNA were isolated with RNase elution and subjected analyzed downstream analysis. Isolated proteins were analyzed by mass spectrometry or Western blotting and chromatin DNA was used for q-PCR, deep sequencing。



一、关于lncRNA在细胞中干扰后接质谱研究的实例


    1.附件文献1研究了lncRNA MALAT1在结肠癌增值和侵袭迁移中的意义,并探讨了机制。研究模式为lncRNA A通过蛋白对C疾病的D表型的影响及机制,为经典研究思路。
    2.文献在lncRNA干扰后进行2D电泳,后接maldi-tof-tof的方法鉴定差异蛋白。是质谱鉴定应用的实例。
    3.文献中2D方法重复性差,可鉴定的差异蛋白数量较少,所获得的蛋白质组学数据难易进行进一步的分析,且这可能是制约该文献影响因子的原因之一。

     基于以上原因,建议进行同类研究的时候,用iTRAQ或者label-free的方法探索lncRNA干扰或者过表达后细胞系的蛋白质组差异,无论对获得的数据数量、质量,甚至经费和时间的支出,都不失为合理之选。




二、lncRNA与蛋白质互做的研究方法:


    附件文献2是近期发表的一篇综述,系统讲解了研究未知功能的lncRNA与蛋白质相互作用机制的方法。其中质谱技术(MS)在 RNA‑centric approach to dissecting lncRNA–protein interactions 相关的研究方法中占有举足轻重的地位。


文献1:MALAT1 promotes colorectal cancer cell proliferation/migration/invasion via PRKA kinase anchor protein 9.
     Our previous studies have shown that the 3' end of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in colorectal cancer(CRC) cell proliferation and migration/invasion in vitro. The role and mechanism of MALAT1 in CRC metastasis in vivo, however, remain largely unknown. In the present study, we found that MALAT1 was up-regulated in human primary CRC tissues with lymph node metastasis. Overexpression ofMALAT1 via RNA activation promoted CRC cell proliferation, invasion and migration in vitro, and stimulated tumor growth and metastasis in mice in vivo. Conversely, knockdown of MALAT1 inhibited CRC tumor growth and metastasis. MALAT1 regulated at least 243 genes in CRC cells in a genome-wide expression profiling. Among these genes, PRKA kinase anchor protein 9 (AKAP-9) was significantly up-regulated at both mRNA and proteinlevels. AKAP-9 was highly expressed in CRC cells with metastatic potential and human primary CRC tissues with lymph node metastasis, but not in normal cells or tissues. Importantly, knockdown of AKAP-9 blocked MALAT1-mediated CRC cell proliferation, migration and invasion. These data indicate that MALAT1 may promote CRC tumor development via its target protein AKAP-9.


      免费全文链接:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268411/


文献2:Unveiling the hidden function of long non-coding RNA by identifying its major partner-protein.
      Tens of thousands of long non-coding RNAs (lncRNAs) have been discovered in eukarya, but their functions are largely unknown. Fortunately, lncRNA-protein interactions may offer details of how lncRNAs play important roles in various biological processes, thus identifying proteins associated with lncRNA is critical. Here we review progress of molecular archetypes that lncRNAs execute as guides, scaffolds, or decoys for protein, focusing on advantages, shortcomings and applications of various conventional and emerging technologies to probe lncRNAs and protein interactions, including protein-centric biochemistry approaches such as nRIP and CLIP, and RNA-centric biochemistry approaches such as ChIRP, CHART and RAP. Overall, this review provides strategies for probing interactions between lncRNAs and protein.
      免费全文链接:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618879/

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邦菲生物——国内首推Orbitrap Fusion™蛋白质组快速鉴定服务
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 楼主| 发表于 2016-1-29 11:59:25 | 显示全部楼层
登陆网页版可直接下载文献全文
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 楼主| 发表于 2016-1-29 11:59:40 | 显示全部楼层
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发表于 2016-1-29 12:01:34 微信发帖 | 显示全部楼层
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发表于 2016-1-29 12:12:59 微信发帖 | 显示全部楼层
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发表于 2016-1-29 18:47:05 | 显示全部楼层

登陆网页版可直接下载文献全文.,菲比?
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发表于 2016-1-29 23:53:57 微信发帖 | 显示全部楼层
blackcarp 发表于 2016-1-29 18:47
登陆网页版可直接下载文献全文.,菲比?

没有菲币的也提供了PMC的链接啊~
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