Wednesday, August 11, 2010

Ribo-Zero rRNA removal and gene expression analysis of fragmented RNA

A significant advantage of the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) is that it can remove virtually all the rRNA, even from degraded total RNA samples. We examined correlation of gene expression between RNA-Seq libraries prepared from intact and fragmented RNA samples treated with the Ribo-Zero Kit, as well as a competitive kit.

Intact and partially fragmented Universal Human Reference RNA (UHRR) samples (2 x 2.5 µg each) were treated with either the Ribo-Zero Kit or a competitive rRNA removal kit. The respective rRNA-depleted samples were pooled and, for each, RNA-Seq libraries were prepared in triplicate from the equivalent of 1 µg total RNA, using a random-primed cDNA synthesis method. Replicates of the respective RNA-Seq libraries were pooled and sequencing was performed using an Illumina GAIIx sequencer with 36-nt reads. The data were analyzed using Illumina’s Pipeline Eland_rna Module and CASAVA Software.

We observed a higher correlation (R2 = 0.9396) in genes detected between intact and fragmented Ribo-Zero rRNA-depleted RNA-Seq libraries (A) compared to the corresponding RNA-Seq libraries prepared using the competitive kit (R2 = 0.8940) (B). Also, an additional 1,016 genes were mapped (with ≥10 reads) for the Ribo-Zero RNA-Seq libraries, indicating better coverage of transcripts with reduced rRNA background.

A. Ribo-Zero rRNA-depleted RNA-Seq libraries

B. Competitor rRNA-depleted RNA-Seq libraries

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