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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.
Highlights
MicroRNAs are a class of small, non-coding RNA species that are broadly expressed in almost all eukaryotes
Quantitative real-time reverse transcription PCR, microarray and small RNA cloning followed by deep sequencing, have been developed for genome-wide Micro RNA (miRNA) profiling
Characterization of systematic bias in current miRNA-Seq method Since the original small RNA cloning technique was developed for de novo miRNA discovery, it has played an instrumental role in uncovering the fascinating world of small RNAs that includes miRNAs [24,25,26], piwi-interacting RNAs [27,28] and endogenous small-interfering RNAs [29,30]
Summary
MicroRNAs (miRNAs) are a class of small (approximately 20 to 22 nucleotides), non-coding RNA species that are broadly expressed in almost all eukaryotes They regulate diverse biological processes by targeting a large number of protein-coding mRNA transcripts [1]. Adding to the complexity of miRNA-mediated regulation, Three major platforms, quantitative real-time reverse transcription PCR (qRT-PCR), microarray and small RNA cloning followed by deep sequencing (miRNA-Seq), have been developed for genome-wide miRNA profiling. Both qRT-PCR and microarray platforms are hybridization-based techniques suitable only for known miRNAs and are unable to provide single-nucleotide resolution for miRNA detection. A highly effective method for genome-wide miRNA quantification by deep sequencing has yet to be successfully developed
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