Hi Katharina,

I know this problem. Both the rRNA depletion systems of Epicentre and Invitrogen often result in only minute amounts of mRNA - very nicely rRNA depleted, but sometimes simply too little material for downstream analyses. This is even more problematic in a host-symbiont situation, where frequently in the harvested material the (e.g. bacterial) symbiont is in much lower amounts than the host cells. However, I have had good experience with an alternative to rRNA depletion: I have successfully used the Epicentre mRNA-ONLY Prokaryotic/ or Eukaryotic mRNA Isolation method for the parallel isolation of bacterial and insect mRNA. This method is based on a 5´-Phosphate-Dependent Exonuclease, an exonuclease that digests RNA having a 5´ monophosphate - which ribosomal rRNAs (both eukaryotic and prokaryotic) have, but not mRNAs. Although you will subsequently have more rRNA "contamination" compared to the rRNA depletion, you will have far less losses. With the resulting material you can then simply run a poly(A) enrichment, to pull out the eukaryotic (poly(A)+) mRNAs from the mRNA pool, while the leftover material will contain your prokaryotic mRNAs (and mRNA fragments, of course).

Hope this might help.

Best regards,

Heiko

I think your best bet from two different organisms would be to use DSN from evrogen. The protocol relies on reannealing rates to reduce rRNA in cDNA library. Based on experience you will have to optimeize the annealing time but we have had a high success rate using this reagent.

http://www.evrogen.com/products/DSN/DSN.shtml

I have experience with only 1 species-specific purification (mammalian cells) and here I had like 5-10% of the starting matter remaining. I guess, you could have a technical problem, rather than a conceptual (MULTIPLEXING).

1. Keep the hybridized rRNA-bead fraction from first step and elute bound RNA (rRNA) which should be of excellent quality, still.

2. Just do a control with either 1 set of beads.

3. Be sure, not to overdry beads or columns.

4. Follow very strictly RNA workflows (use double gloves and discard the upper after each step, work under a PCR hood or equivalent, cleanest water ...).

Hope this helps!

Hi Katharina,

I know this problem. Both the rRNA depletion systems of Epicentre and Invitrogen often result in only minute amounts of mRNA - very nicely rRNA depleted, but sometimes simply too little material for downstream analyses. This is even more problematic in a host-symbiont situation, where frequently in the harvested material the (e.g. bacterial) symbiont is in much lower amounts than the host cells. However, I have had good experience with an alternative to rRNA depletion: I have successfully used the Epicentre mRNA-ONLY Prokaryotic/ or Eukaryotic mRNA Isolation method for the parallel isolation of bacterial and insect mRNA. This method is based on a 5´-Phosphate-Dependent Exonuclease, an exonuclease that digests RNA having a 5´ monophosphate - which ribosomal rRNAs (both eukaryotic and prokaryotic) have, but not mRNAs. Although you will subsequently have more rRNA "contamination" compared to the rRNA depletion, you will have far less losses. With the resulting material you can then simply run a poly(A) enrichment, to pull out the eukaryotic (poly(A)+) mRNAs from the mRNA pool, while the leftover material will contain your prokaryotic mRNAs (and mRNA fragments, of course).

Hope this might help.

Best regards,

Heiko

Since total RNA is >= 80% rRNA your yield does not sound awful (only a few percent is mRNA). As an alternative to the kit method could you try using puromycin and high monovalent salt to dissociate the ribosomal subunits? Then spin the sample on a sucrose cushion in an ultracentrifuge, this will pellet the subunits (but not the mRNAs).

Dear Katharina:

Heike´s answer will be an excellent option. Otherwise you could try using Illumna´s Ribo-Zero Epidemiology, which depletes both eukariotic and prokariotic rRNA, though it is designed for Human/Mouse/Rat and bacteria.

I have tried the above kit with a sample from rumen which contains both prokariotic and eukariotic organisms. I still have to confirm depletion rates by qPCR but from the initial 2.5 microg I recovered around 15-20% of depleted RNA.