【求助】弱问一句，如何给提取的RNA加A尾
丁香园论坛
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如何给提取的RNA加A尾（poly A）
先问一下，你有何目的？
如果是真核的，提的RNA，一般都带polyA，
如果是原核的，那你要加A做什么？
如果是真核的，提的RNA，一般都带polyA，
如果是原核的，那你要加A做什么？
我提的是血清的RNA 但是后期要做microRNA的qRT-PCR
请参考下面这篇文献：
PNAS published ahead of print July 30, 2008,doi:10.1073/pnas.0804549105
Circulating microRNAs as stable blood-based markers for cancer detection
Patrick S. Mitchell†,‡, Rachael K. Parkin†,‡, Evan M. Kroh†,‡, Brian R. Fritz†,§, Stacia K. Wyman†, Era L. Pogosova-Agadjanyan¶, Amelia Peterson†, Jennifer Noteboom‖, Kathy C. O'Briant††, April Allen††, Daniel W. Lin‖,††,‡‡, Nicole Urban††, Charles W. Drescher††, Beatrice S. Knudsen††, Derek L. Stirewalt¶, Robert Gentleman††, Robert L. Vessella‖,‡‡, Peter S. Nelson†,¶, Daniel B. Martin†,§§, and Muneesh Tewari†,¶,¶¶
Abstract
Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
PNAS published ahead of print July 30, 2008,doi:10.1073/pnas.0804549105
Circulating microRNAs as stable blood-based markers for cancer detection
Patrick S. Mitchell†,‡, Rachael K. Parkin†,‡, Evan M. Kroh†,‡, Brian R. Fritz†,§, Stacia K. Wyman†, Era L. Pogosova-Agadjanyan¶, Amelia Peterson†, Jennifer Noteboom‖, Kathy C. O'Briant††, April Allen††, Daniel W. Lin‖,††,‡‡, Nicole Urban††, Charles W. Drescher††, Beatrice S. Knudsen††, Derek L. Stirewalt¶, Robert Gentleman††, Robert L. Vessella‖,‡‡, Peter S. Nelson†,¶, Daniel B. Martin†,§§, and Muneesh Tewari†,¶,¶¶
Abstract
Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
gaoyongxin wrote:
我提的是血清的RNA 但是后期要做microRNA的qRT-PCR
血清中的小RNA，并不用加A尾，然后进行RT，而是有一种stem-loop进行逆转的。
所以每个小RNA都做一个独立的逆转，再用上游的特异性引物和下游的通用引物进行qPCR
我也知道有茎环，但是不知道哪里有合成的，并且经费有限，所以才想着加A尾的
gaoyongxin wrote:
我也知道有茎环，但是不知道哪里有合成的，并且经费有限，所以才想着加A尾的
上海生工，英俊。好像东北的宝生物都有合成。
自己给出序列，让他合成，自己拿回来退火即可。呵呵
60块钱能买5OD左右，用几千次没问题吧
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