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

Zhaojie Zhang; Jerome E. Lee; Kent Riemondy; Emily M. Anderson; Rui Yi

doi:10.1186/gb-2013-14-10-r109

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

Zhaojie Zhang, Jerome E. Lee, Kent Riemondy, Emily M. Anderson, Rui Yi^*

^*Corresponding author for this work

Pathology

Research output: Contribution to journal › Article › peer-review

43 Scopus citations

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.

Original language	English (US)
Article number	R109
Journal	Genome biology
Volume	14
Issue number	10
DOIs	https://doi.org/10.1186/gb-2013-14-10-r109
State	Published - Oct 7 2013

ASJC Scopus subject areas

Ecology, Evolution, Behavior and Systematics
Genetics
Cell Biology

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10.1186/gb-2013-14-10-r109

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title = "High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing",

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.",

author = "Zhaojie Zhang and Lee, {Jerome E.} and Kent Riemondy and Anderson, {Emily M.} and Rui Yi",

note = "Funding Information: We thank D Wang for discussion and providing total RNAs from heart and brain and other members of the Yi lab for suggestions; M Han and B Kudlow for discussion and providing total RNAs from worm; N Lau, S Bradley, W Regan, M Rosbash and J Huntley for reagents and Illumina sequencing; R Dowell for advice on bioinformatics analysis; T Blumenthal for reading the manuscript. This publication was made possible by a start-up fund provided by the University of Colorado and a grant from the NIH (AR059697) to RY and awards from the American Cancer Society to RY and JEL.",

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AU - Riemondy, Kent

AU - Anderson, Emily M.

AU - Yi, Rui

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N2 - 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.

AB - 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.

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

Abstract

ASJC Scopus subject areas

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