Small RNA修飾芯片

  • 簡介
  • 挑戰及解決方案
  • 數據庫
  • 結果展示
  • Arraystar small RNA修飾芯片技術服務在單張芯片可定量miRNA,pre-miRNA和tRNA衍生的small RNA(tsRNA,包括tRF&tiRNA)的堿基修飾。可檢測的修飾包括:8-氧代鳥嘌呤(o8G),7-甲基鳥苷(m7G),N6-甲基腺苷(m6A),假尿苷(Ψ)或5-甲基胞苷(m5C)。 


    芯片優點

    • 能夠檢測及定量多種small RNA上修飾:包括o8G,m7G,m6A,Ψ或m5C

    • 能夠檢測多種small RNA:包括miRNA,pre-miRNA和tsRNAs(tRF&tiRNA)

    • 金標準準確定量small RNA的修飾: 直接RNA末端標記,避免了測序建庫過程中因修飾導致的cDNA延伸中止的情況,可確保對small RNA修飾定量的高保真性。

    • 高靈敏度檢測低水平small RNA的修飾:克服二代測序的局限性,對低表達或低修飾水平的small RNA分析具有出色的分析靈敏度。

    • 所需樣品量少,總RNA量可低至1 µg。


    Arraystar Small RNA修飾芯片列表

    服務名稱 可檢測的修飾* 描述 規格
    Arraystar Human Small RNA 修飾芯片 O8G/m7G/m6A/Ψ/m5C 定量miRNA,pre-miRNA, & tsRNA修飾 8 x 15K
    Arraystar Mouse Small RNA 修飾芯片 O8G/m7G/m6A/Ψ/m5C 定量miRNA,pre-miRNA, & tsRNA修飾 8 x 15K

    單張芯片5修飾選擇一種進行檢測。



  • Small RNA修飾高通量篩選麵臨的挑戰及解決方案

           盡管測序已用於small RNA高通量篩選,但RNA修飾對測序定量的影響仍被嚴重忽視。RNA上多種修飾(m1A,m3C和m1G等)會幹擾測序建庫過程中的逆轉錄,因此small RNA-seq對small RNA修飾的定量是不準確的,特別是對small RNA上的修飾。 例如,Small RNA-seq大多偏向檢測18nt的3’tsRNA,而Northern blot主要檢測到的是22nt的同工型3’tsRNA。這是由於TUC存在m1A,會抑製逆轉錄酶進行逆轉錄。大多數small RNA測序數據是從上述文庫構建方法中獲得的,因此,對於有修飾的small RNA,這些數據可能產生誤導。

           同樣,small RNA-seq需要多個PCR擴增步驟,這會導致明顯的定量偏差及不準確,因此需要使用獨立正交方法。

           事實上,研究修飾的測序方法需要大量的樣本(總RNA> 100 ug),這樣對樣本量有限的研究會產生極大的限製。

           此外,small RNA測序通常使用Reads Per Million(RPM)進行標準化,來表示樣品中RNA的相對豐度。 然而,RPM取決於樣品中small RNA的組成。 一個small RNA的RPM的變化將影響所有其它small RNA的值,即使它們的絕對表達水平沒有改變。

           因此,就需要克服基於測序方法的局限,開發非測序技術,以更高的靈敏度和準確性來鑒定和定量small RNA的修飾譜。


    定量small RNA轉錄後修飾的技術

    Arraystar small RNA修飾芯片技術(圖1)將small RNA芯片與RNA免疫沉澱(RIP)進行整合,可在一張芯片上同時檢測修飾及未修飾small RNA水平,為修飾對small RNA(包括miRNA,pre-miRNA和tRF&tiRNA)的調控提供重要信息,

    圖1. Arraystar small RNA修飾芯片技術,分別鑒定和定量small RNA轉錄後修飾,分別為o8G,m7G,m6A,Ψ和m5C。使用特異性抗體通過免疫沉澱富集修飾的small RNA後,使用Arraystar small RNA修飾芯片進行鑒定和定量。



    參考文獻

    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 修飾芯片 V1.0

    探針總數 14,706
    探針設計策略 整個探針由5’cap區, small RNA特異性區和3’linker區組成。
    探針結合位點 5-p-miRNA 和 5'tsRNA: small RNA的3’區域
    3-p-miRNA 和 3'tsRNA: small RNA的5’區域
    Pre-miRNA: pre-miRNA的頸環區域設計
    探針特異性 small RNA特異性
    miRNA數目 2,628 (1,319個5-p-miRNAs, 1,309個3-p-miRNAs)
    pre-miRNAs數目 1,745
    tsRNAs數目 5,128
    Small RNA來源數據庫 miRNA: miRBase (v22)
    pre-miRNA: miRBase (v22)
    tsRNA: tRFdb, GtRNADb (更新至18.1 2019.08)
    文獻: 公開發表的文獻至 2019 [1-40]
    芯片規格 8 x 15K


    Arraystar Mouse small RNA 修飾芯片 V1.0

    探針總數 14,895
    探針設計策略 整個探針由5’cap區, small RNA特異性區和3’linker區組成。
    探針結合位點 5-p-miRNA 和 5'tsRNA: small RNA的3’區域
    3-p-miRNA 和 3'tsRNA: small RNA的5’區域
    Pre-miRNA: pre-miRNA的頸環區域設計
    探針特異性 small RNA特異性
    miRNA數目 1949 (966個5-p-miRNAs, 983個3-p-miRNAs)
    pre-miRNAs數目 1,122
    tsRNAs數目 1,809
    Small RNA來源數據庫 miRNA: miRBase (v22)
    pre-miRNA: miRBase (v22)
    tsRNA: tRFdb, GtRNADb (更新至18.1 2019.08)
    文獻: 公開發表的文獻至 2019 [1-40]
    芯片規格 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]


  • 數據分析包括可直接使用的關鍵數據、豐富的注釋信息和出版級別的圖形。

    差異修飾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): 基於Cy5標記的m7G-IP RNA的初始信號值得到的log2轉換的標準化後的組平均值。

    Treated, Control:  實驗組和對照組

    FC:  兩組比較的差異倍數

    P:  t test檢驗分析的統計學差異的p值

    Regulation: 兩組比較的上調或者下調

    Group m7G %modified miRNA: m7G修飾的miRNA組平均百分比

    miRNA_family: 具有相同的種子序列的miRNA家族

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


    差異修飾的miRNA,pre-miRNA和tsRNA(tRF&tiRNA)的分層聚類熱圖



    圖1.差異修飾small RNA的分層聚類熱圖。 修飾的RNA水平由左上方小圖中紅藍色色標表示。頂部樹狀圖顯示了樣品之間修飾圖譜相對接近度。組別由熱圖上方的色條表示。