small rna修饰芯片

  • 简介
  • 挑战及凯发app的解决方案
  • 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。可检测的修饰包括: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,避免了测序建库过程中因修饰导致的cdna延伸中止的情况,可确保对small rna修饰定量的高保真性。

    • 高灵敏度检测低水平small rna的修饰:克服二代测序的局限性,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凯发app的解决方案

           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。