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Open Access Highly Accessed Methodology article

New methods for next generation sequencing based microRNA expression profiling

Henk PJ Buermans1*, Yavuz Ariyurek2, Gertjan van Ommen1, Johan T den Dunnen12 and Peter AC 't Hoen1

Author Affiliations

1 Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands

2 Leiden Genome Technology Center, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands

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BMC Genomics 2010, 11:716  doi:10.1186/1471-2164-11-716

Published: 20 December 2010

Abstract

Background

MicroRNAs are small non-coding RNA transcripts that regulate post-transcriptional gene expression. The millions of short sequence reads generated by next generation sequencing technologies make this technique explicitly suitable for profiling of known and novel microRNAs. A modification to the small-RNA expression kit (SREK, Ambion) library preparation method for the SOLiD sequencing platform is described to generate microRNA sequencing libraries that are compatible with the Illumina Genome Analyzer.

Results

High quality sequencing libraries can successfully be prepared from as little as 100 ng small RNA enriched RNA. An easy to use perl-based analysis pipeline called E-miR was developed to handle the sequencing data in several automated steps including data format conversion, 3' adapter removal, genome alignment and annotation to non-coding RNA transcripts. The sample preparation and E-miR pipeline were used to identify 37 cardiac enriched microRNAs in stage 16 chicken embryos. Isomir expression profiles between the heart and embryo were highly correlated for all miRNAs suggesting that tissue or cell specific miRNA modifications do not occur.

Conclusions

In conclusion, our alternative sample preparation method can successfully be applied to generate high quality miRNA sequencing libraries for the Illumina genome analyzer.