Limitations and Possibilities of small RNA Digital Gene Expression Profiling

474 | VOL.6 NO.7 | JULY 2009 | nature methods

To the Editor: High-throughput sequencing (HTS) has proven
to be an invaluable tool for the discovery of thousands of
microRNA genes across multiple species1,2. At present, the
throughput of HTS platforms is sufficient to combine discovery
with quantitative expression analysis allowing for digital gene
expression (DGE) profiling3. We observed that methods for small
RNA DGE profiling are strongly biased toward certain small
RNAs, preventing the accurate determination of absolute numbers
of small RNAs. The observed bias is largely independent of
the sequencing platform but strongly determined by the method
used for small RNA library preparation. However, as the biases are
systematic and highly reproducible, DGE profiling is suited for
determining relative expression differences between samples.
We generated duplicate small RNA libraries using three librarypreparation
methods (poly(A) tailing4, modban adaptor (IDT)
ligation5 and Small RNA Expression kit (SREK; Ambion)) from
a single sample (rat brain) and sequenced these on Roche 454,
AB SOLiD and traditional capillary dideoxy sequencing platforms
(Supplementary Fig. 1, Supplementary Note and Supplementary
Methods). To assess the impact of the library-preparation method
and sequencing platform, we focused on the distribution of
known rat 5′ and 3′ microRNA sequences (miRBase v11.0; ref. 6).

Authors: Sam E V Linsen, Elzo de Wit, Georges Janssens, Sheila Heater, Laura Chapman, Rachael K Parkin, Brian Fritz, Stacia K Wyman, Ewart de Bruijn, Emile E Voest, Scott Kuersten, Muneesh Tewari & Edwin Cuppen

Citation: Limitations and possibilities of small RNA digital gene expression profiling

Linsen et al. Nature Methods 6, 474-476 (30 June 2009) doi:10.1038/nmeth0709-474 correspondence

Link: http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-474.html

This correspondence describes a comparison of 3 library methods for small RNA DGE (Digital Gene Expression) using ModBan (Illumina), SOLiD™ Small RNA Expression Kit (SREK) and a polyA tailing method.

Deixe uma resposta

Preencha os seus dados abaixo ou clique em um ícone para log in:

Logotipo do WordPress.com

Você está comentando utilizando sua conta WordPress.com. Sair / Alterar )

Imagem do Twitter

Você está comentando utilizando sua conta Twitter. Sair / Alterar )

Foto do Facebook

Você está comentando utilizando sua conta Facebook. Sair / Alterar )

Foto do Google+

Você está comentando utilizando sua conta Google+. Sair / Alterar )

Conectando a %s

%d blogueiros gostam disto: