Wednesday, October 27, 2010

rRNA removal from Drosophila total RNA

We have received many inquiries from customers regarding the use of  the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) with RNA from nonmammalian organisms. Although we have not tested this kit with a wide range of genera and species, it will be suitable for many organisms whose rDNA genes exhibit a high degree of homology to human rDNA. However, the rRNA removal efficiency will vary, depending on the degree of homology.

One of our customers, Dr. Dominik Handler at the Institute of Molecular Biotechnology GmbH, Austria, examined the use of the Ribo-Zero (H/M/R) Kit with Drosophila RNA. He compared the amount of rRNA depletion with the Ribo-Zero kit and a competitive kit by qPCR after one and two rounds of rRNA removal.

qPCR analysis of rRNA depletion (click to enlarge figure). Total RNA samples were treated  with one or two rounds of the Ribo-Zero kit (RZ) or a competitive kit (R-), and fold depletion calculated using qPCR with primers to multiple regions of the indicated rRNAs, after normalization to rp49 mRNA. Nitric oxide synthase (nos) mRNA was used as an internal control.

In Drosophila, as in many diptera, the 28S rRNA is processed into two parts (a “left” and “right” arm). After two rounds of rRNA removal, Dr. Handler observed depletion of the left arm by 31,000X and the right arm by 600X. Overall, the Ribo-Zero kit performed substantially better than the competitive kit.

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