瓦力棋牌(中国)

AGO APP-seq /AGO APP芯片

  • 简介
  • 技术优势
  • 实验原理和流程
  • 数据库
  • 结果展示

  •             Argonaute (包含AGO1-4)蛋白是组成RISCs(RNA-induced silencing complex )复合物的主要成员。其经典功能是在细胞质中装载miRNA形成RISC复合物，顺利获得序列互补配对结合靶基因mRNA 3’UTR，促进mRNA降解或蛋白翻译抑制。AGO蛋白-smallRNA的复合物可以分泌到体液中并稳定存在，作为潜在的疾病诊断的生物标志物。

               AGO APP(Affinity Purification by Peptides) smallRNA测序利用特异性结合AGO1-4蛋白的T6B-coupled GST beads富集结合AGO1-4的smallRNA，包括miRNA、tRF&tiRNA等，进行smallRNA测序筛选，从而取得结合AGO蛋白发挥调控功能的smallRNA。

    AGO APP smallRNA芯片服务利用特异性结合AGO1-4蛋白的T6B肽段偶联GST磁珠富集结合AGO蛋白的smallRNA，包括miRNA、tRF&tiRNA、Agotron等，进行smallRNA芯片筛选，从而取得结合AGO蛋白调控RNA稳定性和翻译的smallRNA。




    图1. 血浆外泌体之外结合AGO2稳定存在的miRNA[2]



  • u  AGO APP可同时富集AGO1/2/3/4蛋白结合的smallRNA进行检测；

    u  AGO APP使用的T6B-coupled beads与Ago亲和性高，且检测物种更广泛（从昆虫到人都可检测）；

    u  AGO APP smallRNA芯片能检测miRNA、tRF&tiRNA、Agotron等多种smallRNA，覆盖范围广；

    u  芯片探针检测特异性高，操作简单，样本需求量少。

     

    参考文献：

    [1] Iwakawa HO, et al. Mol Cell. 2022. PMID: 34942118

    [2] Arroyo, Jason D et al. PNAS. 2011. PMID: 21383194

  • 实验原理

    AGO APP(Affinity Purification by Peptides) smallRNA芯片利用特异性结合AGO1-4蛋白的T6B-coupled GST beads富集结合AGO1-4的smallRNA[7]，包括miRNA、tRF&tiRNA、Agotron等，进行AGO APP smallRNA芯片筛选，从而取得结合AGO蛋白发挥调控功能的smallRNA（对于AGO结合的蛋白也可同时进行质谱检测）。

    样本用量：细胞用量>2*10^7个；组织>50mg；血浆血清>2ml。

     

    实验流程

    1.细胞/组织裂解；

    2.AGO APP富集结合的smallRNA；

    3.SmallRNA 3’去磷酸化，DMSO变性，Cy3C标记；

    4.与瓦力棋牌(中国) smallRNA芯片杂交；

    5.扫描芯片，进行数据分析；

    6.给予结果报告。

  • 瓦力棋牌(中国)  Human AGO APP smallRNA芯片V1.0



    References

    1. Hansen, T., Venø, M., Jensen, T. et al. Argonaute-associated short introns are a novel class of gene regulators. Nat Commun 7, 11538 (2016). [PMID: 27173734]
    2. Guzzi N et al: Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells. Cell 2018, 173(5):1204-1216 e1226.[PMID: 29628141]
    3. Keam SP et al: The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells. Nucleic Acids Res 2014, 42(14):8984-8995.[PMID: 25038252]
    4. Keam SP, Sobala A, Ten Have S, Hutvagner G: tRNA-Derived RNA Fragments Associate with Human Multisynthetase Complex (MSC) and Modulate Ribosomal Protein Translation. J Proteome Res 2017, 16(2):413-420.[PMID: 27936807]
    5. Zhang X et al: IL-4 Inhibits the Biogenesis of an Epigenetically Suppressive PIWI-Interacting RNA To Upregulate CD1a Molecules on Monocytes/Dendritic Cells. J Immunol 2016, 196(4):1591-1603.[PMID: 26755820]
    6. Honda S et al: The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells. Nucleic Acids Res 2017, 45(15):9108-9120.[PMID: 28645172]
    7. Cole C et al: Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. RNA 2009, 15(12):2147-2160.[PMID: 19850906]
    8. Sobala A, Hutvagner G: Small RNAs derived from the 5' end of tRNA can inhibit protein translation in human cells. RNA Biol 2013, 10(4):553-563.[PMID: 23563448]
    9. Lee YS, Shibata Y, Malhotra A, Dutta A: A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev 2009, 23(22):2639-2649.[PMID: 19933153]
    10. Huang B et al: tRF/miR-1280 Suppresses Stem Cell-like Cells and Metastasis in Colorectal Cancer. Cancer Res 2017, 77(12):3194-3206.[PMID: 28446464]
    11. Kuscu C et al: tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner. RNA 2018, 24(8):1093-1105.[PMID: 29844106]
    12. Kim HK et al: A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature 2017, 552(7683):57-62.[PMID: 29186115]
    13. Kim HK et al: A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice. Cell Rep 2019, 29(12):3816-3824 e3814.[PMID: 31851915]
    14. Yeung ML et al: Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid. Nucleic Acids Res 2009, 37(19):6575-6586.[PMID: 19729508]
    15. Schorn AJ, Gutbrod MJ, LeBlanc C, Martienssen R: LTR-Retrotransposon Control by tRNA-Derived Small RNAs. Cell 2017, 170(1):61-71 e11.[PMID: 28666125]
    16. Maute RL et al: tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma. Proc Natl Acad Sci U S A 2013, 110(4):1404-1409.[PMID: 23297232]
    17. Ruggero K et al: Small noncoding RNAs in cells transformed by human T-cell leukemia virus type 1: a role for a tRNA fragment as a primer for reverse transcriptase. J Virol 2014, 88(7):3612-3622.[PMID: 24403582]
    18. Falconi M et al: A novel 3'-tRNA(Glu)-derived fragment acts as a tumor-suppressor in breast cancer by targeting nucleolin. FASEB J 2019:fj201900382RR.[PMID: 31560576]
    19. Zhou K et al: A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells. Oncotarget 2017, 8(56):95377-95391.[PMID: 29221134]
    20. Goodarzi H et al: Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 2015, 161(4):790-802.[PMID: 25957686]
    21. Natt D et al: Human sperm displays rapid responses to diet. PLoS Biol 2019, 17(12):e3000559.[PMID: 31877125]
    22. Veneziano D et al: Dysregulation of different classes of tRNA fragments in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2019, 116(48):24252-24258.[PMID: 31723042]
    23. Haussecker D et al: Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 2010, 16(4):673-695.[PMID: 20181738]
    24. Balatti V et al: tsRNA signatures in cancer. Proc Natl Acad Sci U S A 2017, 114(30):8071-8076.[PMID: 28696308]
    25. Cho H et al: Regulation of La/SSB-dependent viral gene expression by pre-tRNA 3' trailer-derived tRNA fragments. Nucleic Acids Res 2019, 47(18):9888-9901.[PMID: 31504775]
    26. Babiarz JE et al: Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev 2008, 22(20):2773-2785.[PMID: 18923076]
    27. Hasler D et al: The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway. Mol Cell 2016, 63(1):110-124.[PMID: 27345152]
    28. Pekarsky Y et al: Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer. Proc Natl Acad Sci U S A 2016, 113(18):5071-5076.[PMID: 27071132]
    29. Liao JY et al: Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3' trailers. PLoS One 2010, 5(5):e10563.[PMID: 20498841]
    30. La Ferlita A et al: Identification of tRNA-derived ncRNAs in TCGA and NCI-60 panel cell lines and development of the public database tRFexplorer. Database (Oxford) 2019, 2019.[PMID: 31735953]
    31. Honda S et al: Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc Natl Acad Sci U S A 2015, 112(29):E3816-3825.[PMID: 26124144]
    32. Donovan J, Rath S, Kolet-Mandrikov D, Korennykh A: Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery. RNA 2017, 23(11):1660-1671.[PMID: 28808124]
    33. Hanada T et al: CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 2013, 495(7442):474-480.[PMID: 23474986]
    34. Saikia M et al: Angiogenin-cleaved tRNA halves interact with cytochrome c, protecting cells from apoptosis during osmotic stress. Mol Cell Biol 2014, 34(13):2450-2463.[PMID: 24752898]
    35. Wang Q et al: Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection. Mol Ther 2013, 21(2):368-379.[PMID: 23183536]
    36. Deng J et al: Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism. Mol Ther 2015, 23(10):1622-1629.[PMID: 26156244]
    37. Zhou J et al: Identification of two novel functional tRNA-derived fragments induced in response to respiratory syncytial virus infection. J Gen Virol 2017, 98(7):1600-1610.[PMID: 28708049]
    38. Yang X et al: 5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader. Cell Res 2017, 27(5):606-625.[PMID: 28418038]
    39. Ivanov P et al: Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 2011, 43(4):613-623.[PMID: 21855800]
    40. Ivanov P et al: G-quadruplex structures contribute to the neuroprotective effects of angiogenin-induced tRNA fragments. Proc Natl Acad Sci U S A 2014, 111(51):18201-18206.[PMID: 25404306]
    41. Schaffer AE et al: CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration. Cell 2014, 157(3):651-663.[PMID: 24766810]


  • 1.差异AGO APP富集的smallRNA注释信息表格

     

     

    2.差异AGO APP富集的smallRNA火山图、散点图、聚类图

         


    3.深入数据分析：smallRNA 靶基因预测及靶基因GO/pathway分析