Small RNA修饰芯片

  • 简介
  • 挑战及解决方案
  • 数据库
  • 结果展示
  • Arraystar small RNA修饰芯片技术服务在单张芯片可定量miRNA,pre-miRNA和tRNA衍生的small RNA(tsRNA,包括tRF&tiRNA)的碱基修饰。目前可检测的修饰有:8-氧代鸟嘌呤(o8G),7-甲基鸟苷(m7G),N6-甲基腺苷(m6A),假尿苷(Ψ)或5-甲基胞苷(m5C)。 


    芯片优点

    • 能够检测及定量多种small RNA上修饰:包括o8G,m7G,m6A,Ψ或m5C

    • 能够检测多种small RNA:包括miRNA,pre-miRNA和tsRNAs(tRF&tiRNA)

    • 金标准准确定量small RNA的修饰

          直接RNA末端标记可确保定量的高保真性。

          不会产生逆转录或PCR扩增造成的数据偏倚。

          芯片探针设计可微调探针与靶基因的杂交Tm,以获得高灵敏度和特异性。 

          定量的精确性优于其他方法。

    • 高灵敏度检测低水平small RNA的修饰

    克服small RNA-seq方法局限性,对低表达或低修饰水平的small RNA分析具有出色的分析灵敏度。

    • 所需样品量少,总RNA量可低至1 µg。


    Arraystar Small RNA修饰芯片列表

    服务名称 可检测的修饰* 描述 规格
    Arraystar Human Small RNA 修饰芯片 O8G/m7G/m6A/Ψ/m5C 定量miRNA,pre-miRNA, & tsRNA修饰 8 x 15K
    Arraystar Mouse Small RNA 修饰芯片 O8G/m7G/m6A/Ψ/m5C 定量miRNA,pre-miRNA, & tsRNA修饰 8 x 15K

    单张芯片5修饰选择一种进行检测。



  • Small RNA修饰高通量筛选面临的挑战及解决方案

           尽管测序已用于small RNA高通量筛选,但RNA修饰对测序定量的影响仍被严重忽视。RNA上多种修饰(m1A,m3C和m1G等)会干扰测序建库过程中的逆转录,因此small RNA-seq对small RNA修饰的定量是不准确的,特别是对small RNA上的修饰。 例如,Small RNA-seq大多偏向检测18nt的3’tsRNA,而Northern blot主要检测到的是22nt的同工型3’tsRNA。这是由于TUC存在m1A,会抑制逆转录酶进行逆转录。大多数small RNA测序数据是从上述文库构建方法中获得的,因此,对于有修饰的small RNA,这些数据可能产生误导。

           同样,small RNA-seq需要多个PCR扩增步骤,这会导致明显的定量偏差及不准确,因此需要使用独立正交方法。

           事实上,研究修饰的测序方法需要大量的样本(总RNA> 100 ug),这样对样本量有限的研究会产生极大的限制。

           此外,small RNA测序通常使用Reads Per Million(RPM)进行标准化,来表示样品中RNA的相对丰度。 然而,RPM取决于样品中small RNA的组成。 一个small RNA的RPM的变化将影响所有其它small RNA的值,即使它们的绝对表达水平没有改变。

           因此,就需要克服基于测序方法的局限,开发非测序技术,以更高的灵敏度和准确性来鉴定和定量small RNA的修饰谱。


    定量small RNA转录后修饰的技术

    Arraystar small RNA修饰芯片技术(图1)将small RNA芯片与RNA免疫沉淀(RIP)进行整合,可在一张芯片上同时检测修饰及未修饰small RNA水平,为修饰对small RNA(包括miRNA,pre-miRNA和tRF&tiRNA)的调控提供重要信息,

    图1. Arraystar small RNA修饰芯片技术,分别鉴定和定量small RNA转录后修饰,分别为o8G,m7G,m6A,Ψ和m5C。使用特异性抗体通过免疫沉淀富集修饰的small RNA后,使用Arraystar small RNA修饰芯片进行鉴定和定量。



    参考文献

    1.    Guzzi N et al: Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells. Cell 2018, 173(5):1204-1216 e1226.[PMID: 29628141]

    2.    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]

    3.    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]

    4.    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]

    5.    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]

    6.    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]

    7.    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]

    8.    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]

    9.    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]

    10.  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]

    11.  Kim HK et al: A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature 2017, 552(7683):57-62.[PMID: 29186115]

    12.  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]

    13.  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]

    14.  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]

    15.  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]

    16.  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]

    17.  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]

    18.  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]

    19.  Goodarzi H et al: Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 2015, 161(4):790-802.[PMID: 25957686]

    20.  Natt D et al: Human sperm displays rapid responses to diet. PLoS Biol 2019, 17(12):e3000559.[PMID: 31877125]

    21.  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]

    22.  Haussecker D et al: Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 2010, 16(4):673-695.[PMID: 20181738]

    23.  Balatti V et al: tsRNA signatures in cancer. Proc Natl Acad Sci U S A 2017, 114(30):8071-8076.[PMID: 28696308]

    24.  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]

    25.  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]

    26.  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]

    27.  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]

    28.  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]

    29.  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]

    30.  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]

    31.  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]

    32.  Hanada T et al: CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 2013, 495(7442):474-480.[PMID: 23474986]

    33.  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]

    34.  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]

    35.  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]

    36.  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]

    37.  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]

    38.  Ivanov P et al: Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 2011, 43(4):613-623.[PMID: 21855800]

    39.  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]

    40.  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]

  • Arraystar  Human small RNA 修饰芯片 V1.0

    探针总数 14,706
    探针设计策略 整个探针由5’cap区, small RNA特异性区和3’linker区组成。
    探针结合位点 5-p-miRNA 和 5'tsRNA: small RNA的3’区域
    3-p-miRNA 和 3'tsRNA: small RNA的5’区域
    Pre-miRNA: pre-miRNA的颈环区域设计
    探针特异性 small RNA特异性
    miRNA数目 2,628 (1,319个5-p-miRNAs, 1,309个3-p-miRNAs)
    pre-miRNAs数目 1,745
    tsRNAs数目 5,128
    Small RNA来源数据库 miRNA: miRBase (v22)
    pre-miRNA: miRBase (v22)
    tsRNA: tRFdb, GtRNADb (更新至18.1 2019.08)
    文献: 公开发表的文献至 2019 [1-40]
    芯片规格 8 x 15K


    Arraystar Mouse small RNA 修饰芯片 V1.0

    探针总数 14,895
    探针设计策略 整个探针由5’cap区, small RNA特异性区和3’linker区组成。
    探针结合位点 5-p-miRNA 和 5'tsRNA: small RNA的3’区域
    3-p-miRNA 和 3'tsRNA: small RNA的5’区域
    Pre-miRNA: pre-miRNA的颈环区域设计
    探针特异性 small RNA特异性
    miRNA数目 1949 (966个5-p-miRNAs, 983个3-p-miRNAs)
    pre-miRNAs数目 1,122
    tsRNAs数目 1,809
    Small RNA来源数据库 miRNA: miRBase (v22)
    pre-miRNA: miRBase (v22)
    tsRNA: tRFdb, GtRNADb (更新至18.1 2019.08)
    文献: 公开发表的文献至 2019 [1-40]
    芯片规格 8 x 15K


    References


    1. Guzzi N et al: Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells. Cell 2018, 173(5):1204-1216 e1226.[PMID: 29628141]

    2. 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]

    3. 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]

    4. 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]

    5. 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]

    6. 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]

    7. 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]

    8. 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]

    9. 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]

    10. 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]

    11. Kim HK et al: A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature 2017, 552(7683):57-62.[PMID: 29186115]

    12. 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]

    13. 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]

    14. 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]

    15. 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]

    16. 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]

    17. 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]

    18. 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]

    19. Goodarzi H et al: Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement. Cell 2015, 161(4):790-802.[PMID: 25957686]

    20. Natt D et al: Human sperm displays rapid responses to diet. PLoS Biol 2019, 17(12):e3000559.[PMID: 31877125]

    21. 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]

    22. Haussecker D et al: Human tRNA-derived small RNAs in the global regulation of RNA silencing. RNA 2010, 16(4):673-695.[PMID: 20181738]

    23. Balatti V et al: tsRNA signatures in cancer. Proc Natl Acad Sci U S A 2017, 114(30):8071-8076.[PMID: 28696308]

    24. 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]

    25. 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]

    26. 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]

    27. 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]

    28. 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]

    29. 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]

    30. 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]

    31. 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]

    32. Hanada T et al: CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 2013, 495(7442):474-480.[PMID: 23474986]

    33. 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]

    34. 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]

    35. 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]

    36. 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]

    37. 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]

    38. Ivanov P et al: Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 2011, 43(4):613-623.[PMID: 21855800]

    39. 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]

    40. 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]


  • 数据分析包括可直接使用的关键数据、丰富的注释信息和出版级别的图形。

    差异修饰small RNA列表,包括miRNA,pre-miRNA和tsRNA(tRF&tiRNA)


    MatureID:  成熟miRNA在miRBase 的ID

    Group m7G miRNA level (normalized, log2): 基于Cy5标记的m7G-IP RNA的初始信号值得到的log2转换的标准化后的组平均值。

    Treated, Control:  实验组和对照组

    FC:  两组比较的差异倍数

    P:  t test检验分析的统计学差异的p值

    Regulation: 两组比较的上调或者下调

    Group m7G %modified miRNA: m7G修饰的miRNA组平均百分比

    miRNA_family: 具有相同的种子序列的miRNA家族

    m7G_motif: “RAm7GGT” m7G 的motif基序, R 代表G或A


    差异修饰的miRNA,pre-miRNA和tsRNA(tRF&tiRNA)的分层聚类热图



    图1.差异修饰small RNA的分层聚类热图。 修饰的RNA水平由左上方小图中红蓝色色标表示。顶部树状图显示了样品之间修饰图谱相对接近度。组别由热图上方的色条表示。