- Simple procedure takes less than 2 hours
- Remove >90% of mammalian RNA from complex mixtures of host-bacterial samples
- Works with any bacterial species
- Enables microarray expression analysis with bacterial RNA isolated from host cells
- Seamless integration with MICROBExpress™ Bacterial mRNA Enrichment Kit
Gene expression analysis following host-bacterial interactions
is revolutionizing our understanding of the host response to bacterial
infection. Numerous studies have utilized microarray analysis to follow
host cell transcriptome alterations in response to interactions
with bacteria. However, because no method has existed to isolate bacterial
RNA away from host cell RNA, the corresponding analyses of bacterial transcriptomes
has been impossible. Ambion now introduces a new technology, MICROBEnrich,
that enables whole genome microarray expression analyses of bacterial
RNA after host-bacterial interactions.
Start With Any Total RNA Sample
Although the mass of eukaryotic RNA isolated from
host-bacterial cell mixtures is often far greater than the mass of bacterial
RNA isolated, this complex mixture of total RNAs is the starting material
for the MICROBEnrich procedure. This host-bacterial cell total
RNA mixture can be obtained by a variety of RNA isolation methods, ideally
by Ambion's new RiboPure™-Bacteria
Kit. Other methods including glass fiber filter based methods (such
as Ambion's RNAqueous® Kits)
and phenol based methods (such as TRI Reagent®
and Ambion's ToTALLY RNA™ Kit)
are also appropriate. For microarray analysis and other sensitive applications,
Ambion also recommends a DNase treatment (such as with DNA-free™
Treatment and Removal Reagents) to remove contaminating genomic DNA
from the total RNA prior to the MICROBEnrich procedure.
Remove >90% of Mammalian RNA from Complex
Total RNA Mixtures
MICROBEnrich employs hybridization capture
technology (patent pending) to remove human, mouse, and rat RNA (both
mRNA and rRNA) from complex host-bacterial RNA populations, leaving behind
enriched microbial total RNA. In the first step of the MICROBEnrich
procedure, host-bacterial total RNA is incubated with an optimized mixture
of capture oligonucleotides that bind to the mammalian 18S and 28S rRNAs
and polyadenylated RNAs. Next, the rRNA/oligo nucleotide hybrids and all
polyadenylated mRNAs are removed from the mixture with oligonucleotide-derivatized
magnetic beads. After magnetic separation, the bacterial RNA remaining
in the supernatant and can be precipitated with ethanol. Figure 1 shows
an Agilent 2100 bioanalyzer electropherogram of a complex host-bacterial
RNA population before and after bacterial RNA purification with MICROBEnrich.
The RNA profile indicates nearly complete removal (>95%) of the host
18S and 28S rRNAs.
Removal of Bacterial rRNA
If desired, upon completion of the MICROBEnrich
procedure bacterial mRNA can be purified from the total RNA through the
seamless integration of Ambion's MICROBExpress technology. This
technology enables >95% removal of prokaryotic 16S and 23S rRNAs (Figure
2).
MICROBEnrich Will Work With Any Bacterial
Species
MICROBEnrich was optimized using E.
coli RNA in a background of human, mouse and rat RNA. However, the
kit should perform well with RNA from any bacterial species. Although
many mammalian host species may be compatible with MICROBEnrich,
only human, mouse and rat rRNA sequences were used for the design and
optimization of the capture oligonucleotides.
Increased Sensitivity in Downstream Analyses
Bacterial mRNAs purified with the combined MICROBEnrich
and MICROBExpress Kits serve as better templates than total RNA
for downstream applications and therefore deliver dramatic increases in
sensitivity when used in applications such as array analysis. Figure 3
shows Sigma-Genosys Panorama™ E. coli arrays that
were hybridized with labeled cDNA derived from either a mixed population
of total RNA (E. coli/HeLa cell RNA mixture) or mRNA purified from
the mixture using the MICROBEnrich and MICROBExpress Kits.
The number of transcripts detected with labeled RNA target increases substantially
after bacterial mRNA enrichment using the MICROBEnrich and MICROBExpress
Kits.
Reagents to Process 500 µg of Host Cell
RNA
The MICROBEnrich Kit comes with reagents
capable of depleting 500 µg of host cell RNA (20 rxns x 25 µg
RNA) from a complex host-bacterial RNA mixture. The Kit is scalable, and a single reaction can
process between 10 and 100 µg of total RNA. The Kit includes a control
RNA sample (E. coli and rat liver total RNA) and a detailed Instruction
Manual with tips and troubleshooting advice.
The MICROBEnrich protocol requires the use of a magnetic stand, available separately.
Trademarks, Patents, Licensing
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