Kit from Ambion is used to remove human, mouse and rat RNA from a mix of mammalian and bacterial RNA. It uses a hybridization capture technology to bind to mammalian 18S and 28S ribosomal RNA and polyadenylated messenger RNA. The kit comes complete with capture oligonucleotides, oligo magnetic beads and buffers for bead regeneration. However, it does not come supplied with the magnetic stand, which needs to be purchases separately (cat # AM10026 – single stand and cat # AM10055 – 6 tube stand).
I infect human cells with bacteria and first extract total RNA from the sample using different kits. I then use the Ambion MicrobEnrich Kit to enrich my bacterial RNA. The protocol booklet that is included in the kit says that up to a 100 ìg of starting RNA can be used; however, I have never used more than 50 ug of RNA to start with. This amount gives about 20 ug of bacterial RNA at the end of the procedure.
The total RNA sample is first incubated with oligonucleotides that bind to 18S and 28S rRNA and to the polyadenylated (AAA repeats) tails on mammalian mRNA. The magnetic beads then bind to the bound oligonucleotides. The bead-oligonucleotide-mRNA complexes are removed from the sample by placing the sample on the magnetic stand which pulls the beads to the side of tube. This enables us to collect the enriched bacterial RNA sample. The RNA is then ethanol precipitated to collect and use for further work.
Though the protocol suggests that the minimal starting amount of RNA can be 5 ug, I have never been able to use less than 20 ug of starting material as the amount of bacterial RNA enriched is too little for successful recovery. The kit also comes with regeneration solutions that are used to regenerate the magnetic beads so that they can be used more than once. However, every time I have re-used the beads, the amount of enriched RNA I have recovered has reduced and the chance of degradation has increased.
Another problem is the time the entire procedure takes. Since ethanol precipitation is needed to recover the RNA, this can easily add close to 3 hours to the protocol after the actual enrichment procedure which takes about 2 hours. Also, I have found that RNA can be lost when using ethanol precipitation for recovery and that precipitation can be a problematic when very small amounts of starting material are used. In addition, I have used the enrichment kit twice on the same sample when sufficient enrichment wasn’t achieved. This was probably because the initial sample contained a very large amount of human RNA. Using the kit twice on the same sample caused degradation of the RNA probably due to excessive handling of the RNA. However, the kit usually gives very good quality RNA which has very little or no trace of the mammalian RNA. I am then able to use this RNA for microarray analysis.
The bottom line is that the MicrobEnrich kit has proved to be very efficient in removing mammalian RNA from mixed samples and is still better than any other technique I have used.
MicrobEnrich Kit From Ambion
Removes mammalian RNA, leaving viable bacterial RNA.
Cannot always reuse magnetic beads.
The Bottom Line
Very efficient for isolating bacterial RNA from mixed samples.