High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing
- Equal contributors
1 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, 80305, USA
2 Dharmacon Inc., a wholly owned subsidiary of Thermo Fisher Scientific, Inc., Lafayette, CO, 80026 USA
Genome Biology 2013, 14:R109 doi:10.1186/gb-2013-14-10-r109Published: 7 October 2013
Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.