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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

Additional files

Additional file 1:

Tables S1 to S3 and figures S1 to S3. Table S1: synthetic miRNA library. Complete list of 29 synthetic miRNAs and the primary sequences used in miRNA-Seq studies. Table S2: GC content of synthetic miRNA library. List of 29 synthetic miRNAs used in this study, their corresponding GC content listed as a percentage of total nucleotide number, and their observed cloning frequency listed as a percentage of total mapped miRNA reads. Table S3: oligonucleotides used in this study. List of oligonucleotides used in the ligation optimization and miRNA-Seq experiments. Figure S1: clustered miRNAs 143/145 exhibit differential quantification in a method-specific manner. (A) miRNA-Seq from mouse hair follicle. (B) qRT-PCR quantification of mouse epidermis determined by ΔΔCt method where sno25 serves as the reference gene. Figure S2: DNA oligos can be readily adenylated by MTH (Methanobacterium thermoautotrophicum) RNA ligase. 18% urea-PAGE of 3′ DNA linkers shows shift in electrophoretic mobility corresponding to 5′ adenylation where plus and minus signs indicate the presence or lack of MTH enzyme, respectively. Figure S3: enhanced miRNA-Seq approach is highly correlative for varying amounts of input RNA. Differing amounts of the 29 synthetic miRNA mix were subjected to enhanced miRNA-Seq.

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