作者
Markus Hafner, Neil Renwick, Miguel Brown, Aleksandra Mihailović, Daniel Holoch, Carolina Lin, John TG Pena, Jeffrey D Nusbaum, Pavel Morozov, Janos Ludwig, Tolulope Ojo, Shujun Luo, Gary Schroth, Thomas Tuschl
发表日期
2011/9/1
期刊
Rna
卷号
17
期号
9
页码范围
1697-1712
出版商
Cold Spring Harbor Lab
简介
Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3′ and 5′ ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1–249) and Rnl2(1–249)K227Q, for 3′-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5′-adapter ligation step; RT and PCR steps did not have a significant …
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M Hafner, N Renwick, M Brown, A Mihailović, D Holoch… - Rna, 2011