TGIRT™-III Enzyme
规格:
10 Reactions (TGIRT10)
50 Reactions (TGIRT50)
5 x 50 µl (TGIRT5x50)
10 x 50 µl (TGIRT10x50)
单位定义:
TGIRT的一个单元® -III逆转录酶(RT)活性是酶的使用聚在60℃下聚合1纳摩尔的dNTP的在1分钟(RA)所需要的量/寡(dT)42作为底物。
酶浓度:
200单位/μl
酶储存缓冲液:
20mM Tris-HCl(pH 7.5),500mM KCl,1mM EDTA,1mM DTT,50%甘油
酶属性和新颖活动:
比逆转录逆转录酶更高的热稳定性,持续合成能力和保真度,允许从高度结构化或重度修饰的RNA (例如tRNA)和包含富含GC的重复扩增的RNA合成全长,端对端cDNA 。1-9,12,15,18
新型端对端模板转换活动,可在反转录过程中连接RNA-seq或PCR适配器,并且无需单独的RNA 3'-适配器连接步骤。1这种模板转换活性极大地促进了链特异性RNA-seq文库的构建,而且比使用随机六聚体引物或使用RNA连接酶进行衔接子连接的方法具有更小的偏差。1,7,8
从退火引物合成cDNA。将退火的引物应具有推测的T 米 > 60的直径: C.酶与反应底物混合,在室温下,通过加入dNTP的反应开始30分钟的预温育,建议。新应用的zui宜条件应该通过测试25-450mM NaCl的一系列盐浓度来确定。
建议用于酶的用途:
1.全面的链特异性转录组分析。8
2.全细胞,外泌体,血浆和其他无细胞RNA的RNA-seq。7,8,15
3.分析miRNA,tRNA和其他小的非编码RNA。1-9,12,15
4. RIP-seq,HITS-CLIP,irCLIP和CRAC用于表征RNA-蛋白质相互作用和核糖体分析。1,10,11
5.通过高通量测序鉴定RNA碱基修饰。4,5,13,14
6.使用诸如SHAPE和DMS修饰的方法进行全基因组或靶向RNA结构作图。1,16
7.富含GC重复扩增的逆转录和定量。18
8.长cDNA的合成。1
9.RT-qPCR。1
10.单链DNA-seq。17
11.分析FFPE肿瘤样本(咨询InGex)。
酶的优点:
1.全面的链特异性转录组分析。
核糖核碎片,通用人类参考RNA样品的TGIRT®-seq概括了人类转录物和穗突起的相对丰度,与非链特异性TruSeq v2相比,并且优于链特异性Tru-Seq v3。TGIRT®-seq比TruSeq v3具有更高的链特异性,并消除了TruSeq固有的随机六聚体引发的取样偏差。TGIRT®-seq显示出更加统一的5'至3'基因覆盖范围,并且比TruSeq识别更多的剪接点。TGIRT®-seq能够在与结构化小型ncRNA相同的RNA-seq中同时分析mRNA和lncRNA,包括tRNA,TruSeq数据集基本上不存在tRNA。8
2.全细胞,外泌体,血浆和其他细胞外RNA的RNA-seq。
快速的处理时间(通过PCR步骤对RNA-seq文库构建<5小时); 需要少量的RNA(低ng范围); 包括mRNA和lncRNA以及小ncRNA的全面转录谱,包括tRNA,pre-miRNA和其他结构化小型ncRNA的全长读段; 比常规方法更少的偏差和更大的链特异性。7,8,15
3.通过TGIRT®模板转换的RNA-seq文库构建,如RIP-seq,HITS-CLIP,irCLIP,CRAC,核糖体谱分析。
快速的处理时间(通过PCR步骤对RNA-seq文库构建<5小时); 需要少量的RNA(低ng范围); 不需要RNA连接酶,通过减少步骤中的步骤来减少偏倚并提率。1,10,11
4.比逆转录病毒RT更高的热稳定性,持续合成能力和链置换活性。
使用锚定寡核苷酸(dT)引物,可以构建RNA聚合腺苷酸化的RNA文库,与没有ribodepletion步骤的逆转录病毒RT相比,具有更均匀的5'至3'覆盖范围。1
通过基于毛细管电泳的方法(如SHAPE或DMS结构图)进行RNA结构作图,其显着的读数长度和更少的提前停止时间比逆转录病毒RTs更短。1,16
可以分析含有富含GC的重复扩增的RNA模板。18
能够合成来自tRNA和其他小型结构化/修饰的ncRNA的全长,端对端cDNA,这些逆转录病毒RT难以治疗。4-9,12-15
5.人血浆和大肠杆菌 基因组DNA的ssDNA-seq 。
通过直接在DNA链的3'末端启动DNA合成,同时连接DNA-seq接头而不进行末端修复,拖尾或连接,以更简单的工作流程捕获的DNA末端。能够分析核小体定位,转录因子结合位点,DNA甲基化位点和起源组织。17
参考文献:
- Mohr, S., Ghanem, E., Smith, W., Sheeter, D., Qin, Y., King, O., Polioudakis, D., Iyer, V.R., Hunicke-Smith, S. Swamy, S., Kuersten, S., and Lambowitz, A.M. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958-970, 2013.
- Collins, K. and Nilsen, T. Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology. RNA 19, 1017-1018, 2013.
- Enyeart, P.J., Mohr, G., Ellington, A.D., and Lambowitz A.M. Biotechnological applications of group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis. DNA 5:2, 2014.
- Katibah, G.E., Qin, Y., Sidote, D.J., Yao, J., Lambowitz, A.M. and Collins, K. Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5. Proc. Natl. Acad. Sci., USA, 111, 12025-12030, 2014.
- Shen, P.S., Park, J., Qin, Y., Li, X., Parsawar, K., Larson, M.H., Cox, J., Chen, Y., Lambowitz, A.M., Weissman, J.S., Brandman, O., and Frost, A. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347, 75-78, 2015.
- Zheng, G., Qin, Y., Clark, W.C., Yi, C., He, C., and Lambowitz, A.M. and Pan, T. Efficient and quantitative high-throughput transfer RNA sequencing. Nat. Methods 12, 835-837, 2015.
- Qin,Y., Yao,J., Wu,D., Nottingham, R., Mohr, S, Hunicke-Smith, S., Lambowitz, A.M., High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases. RNA 22, 111-128, 2016.
- Nottingham, R.M., Wu, D.C., Qin, Y., Yao, J., Hunicke-Smith, S., and Lambowitz, A.M. RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase. RNA 22, 597-613, 2016.
- Burke, J.M., Kincaid, R.P., Nottingham, R.M., Lambowitz, A.M., and Sullivan, C.S. DUSP11 activity on tri-phosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady state levels of cellular non-coding RNAs. Genes & Dev. 30, 2071-2092, 2016.
- Zarnegar, B.J., Flynn, R.A., Shen, Y., Do, B.T., Chang, H.Y. and Khavari, P.A. irCLIP platform for characterization of protein-RNA interactions. Nat. Methods 13, 489-492, 2016.
- Haque, N. and Hogg, J.R. Easier, better, faster, stronger: Improved methods for RNA-protein interaction studies, Mol. Cell, 2016 http//dx.doi.org/10.1016/j.molcel.2016.05.019
- Bazzini, A.A., del Viso, F., Moreno-Mateos, M.A., Johnstone, T.G., Vejnar, C.E., Qin, Y., Yao, J., Khokha, M.K., and Giraldez, A.J. Codon identity regulates mRNA stability and translation efficiency during the maternal-to-zygotic transition. EMBO J. 35, 2087-2103, 2016.
- Clark, W.C., Evans, M.E., Dominissini, D., Zheng, G., and Pan, T. tRNA base methylation identification and quantification via high-throughput sequencing. RNA 22, 1771-1784, 2016.
- Liu et al. ALKBH-mediated tRNA demethylation regulates translation. Cell 167, 816-828, 2016,
- Shurtleff, M.J., Yao, J., Qin, Y., Nottingham, R.M., Temoche-Diaz, M., Schekman, R., and Lambowitz, A.M. A broad role for YBX1 in defining the small non-coding RNA composition of exosomes. Proc. Natl. Acad. Sci. U.S.A. 114, 8987-8995, 2017.
- Zubradt, M., Gupta, P., Persad, S., Lambowitz, A.M., Weissman, J.S., and Rouskin, S. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nature Methods 10.1038/nmeth.4057, 2016.
- Wu, D.C., and Lambowitz, A.M. Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching. Scientific Reports 7, 8421, 2017.
- Carrell, S.T., Tang, Z., Mohr, S., Lambowitz, A.M, and Thorton, C.A. Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase. Nucleic Acids Res. 46, e1, 2018.
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