A team led by researchers at the Austrian Academy of Sciences has developed a protocol that includes column-free RNA preparation, ribosomal RNA depletion, RNA-hydrolysis and ds-cDNA synthesis and is compatible with the all of the major NGS platforms used to date.
They demonstrate that:
- Ribo-depleted RNA-Seq is highly reproducible between different sequencing locations, even when using two different ribosomal RNA depletion strategies.
- Template fragmentation by RNA-hydrolysis produces more homogenous gene coverage than cDNA shearing, and that both fragmentation methods lead to under-representation of 5′ and 3′ UTRs.
- The use of similar template preparation protocols is critical for obtaining a comparable transcriptome.
- RNA populations prepared by ribo-depletion allow RNA-Seq to reliably detect both the non-coding and protein-coding transcriptome, and also to identify biologically relevant gene expression differences in both of these RNA types.
Huang R, Jaritz M, Guenzl P, Vlatkovic I, Sommer A, et al. (2011) An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs. PLoS ONE 6(11), e27288. [article]