Drosophila mRNA purification, 18s trouble!

[SeqByChoice]

Hi,

using Dynabeads mRNA purification kit (polyA, LifeTech), on Drosophila total RNA, we are always left with about 11% rRNA. Primarily 18s.
We have tried double and triple purification, re-using beads, using new beads; and with varying dilutions of the RNA and the beads.
The kit normally works beautifully on mouse and human RNA.
Can anybody pls tell us how make it work on Drosophila???

SbC

[Olaf Blue]

Not certain how close, or helpful this is, but a relatively ancient article concerning the presence of 18s rRNA sequences in mRNAs may be useful (included Drosophila embryos):

http://www.pnas.org/content/94/2/422.full.pdf

[cliffbeall]

I am not sure if this is related but mitochondrial rRNA is polyadenylated in Drosophila.

Are you sure its 18S, not smaller? By the way, the 28S in Drosophila is in 2 pieces, so if you look on the bioanalyzer or gel you get 3 peaks really close together in total RNA. PolyA+ RNA is enriched in this mitochondrial thing, that is around 1 kb if I recall.

eg - http://nar.oxfordjournals.org/conten.../9880.full.pdf

[Olaf Blue]

Very well could be as we do note that these sequences are present in other eukaryotic sample types. the citation does mention that the sequences have homology with 18s.

[SeqByChoice]

Hi,
thanks for your good insights in helping me troubleshoot this!
The "raw" total RNA bioanalyzer trace gives three nice peaks: small peak at about 3600nt and the two high peaks at ~2000nt and 1800nt (18s and broken 28s I suppose).
Several rounds of polyA enrichment still leaves approx 11% of the 2000nt peak (no sign of the mitoch 1000nt peak).
Yesterday we decided to "crunch" what we cant solve, and did first a round of RiboMinus and then the polyA enrichment. We are now down to an acceptable 5% of the 2000nt peak.
Actually I just now found a link to someone who has systematically tested how to get rid of the Drosophila rRNA:

https://sites.google.com/site/rnajunkies/sample-preparation/rrna-depletion/drosophila-rrnas-be-gone

However..... RiboMinus is expensive... and a quick calculation shows that its actually in this case cheaper for us to generate 10% more reads than to apply the RiboMinus kit..... What a peculiar revelation!

SbC

[Olaf Blue]

Thanks for the reply and best of luck SbC!

Olaf