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High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing

Zhaojie Zhang1, Jerome E Lee1, Kent Riemondy1, Emily M Anderson2 and Rui Yi1*

Author Affiliations

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

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Genome Biology 2013, 14:R109  doi:10.1186/gb-2013-14-10-r109

Published: 7 October 2013

Abstract

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.