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 |
* 单张芯片可从5种修饰中选择一种进行检测。
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。