High-efficiency RNA cloning enables quantitative measurement of miRNA expression by deep sequencing
Transcriptomics Dataset
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General Information
AccessionGSE47858
TitleHigh-efficiency RNA cloning enables quantitative measurement of miRNA expression by deep sequencing
DescriptionMiRNA-mediated regulation depends on the stoichiometry between miRNAs and their mRNA targets. To decipher dynamic function of this complex layer, it is critical to characterize individual miRNA species within a specific cellular context. Small RNA cloning followed by deep sequencing is uniquely positioned as a genome-wide profiling method to quantify miRNA expression with potentially unlimited dynamic range and provide single-nucleotide resolution for precise miRNA classification and de novo discovery. However, significant biases introduced by RNA ligation steps in the current RNA cloning protocol often lead to inaccurate miRNA quantification by >1000-fold deviation. As a result, it has greatly hindered the broad application of this method. Here we report a highly efficient RNA cloning method that achieves over 90%% efficiency for both 5 and 3 ligations with diverse small RNA substrates. When applied to a pool of either equimolar or differentially mixed synthetic miRNAs, the deviation of the cloning frequency for each miRNA is minimized to less than 2-fold of the anticipated value. By using samples obtained from multiple tissues and cells, we further demonstrate the accurate quantification of miRNA expression over a dynamic range of four orders of magnitude. Our results also reveal that most cistronic miRNAs are expressed at similar levels and, in each cell population, miRNAs repress their cognate targets in a dosage dependent manner. Collectively, our high-efficiency RNA cloning method combining with deep sequencing establishes a cost-effective approach for accurate genome-wide miRNA profiling.
Landing Pagehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47858
RepositoryGene Expression Omnibus
Tools used to analyze the dataset
Dataset Accession
Clustergrammer
Published October 2017
Web-based tool for visualizing and analyzing high-dimensional data as interactive and shareable heatmaps
4645
ARCHS4
Published September 2017
ARCHS4 provides access to gene counts from HiSeq 2000 and HiSeq 2500 platforms for human and mouse experiments from GEO ...
4645
Canned Analyses generated with the dataset
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Interactive heatmap visualization of RNA-seq dataset GSE47858
ARCHS4, Clustergrammer
Highly interactive web-based heatmap visualization of the top 500 most variable genes in t...
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