MICROBEnrich™
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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 Ambion's RNAwiz™ and the ToTALLY RNA™ Kit) are also appropriate. For microarray analysis and other sensitive applications, Ambion also recommends a DNase treatment (such as with DNAfree™ 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.
Figure 1. RNA Before and After Enrichment with MICROBEnrich™ Kit. Rat liver total RNA (25 µg) and E. coli total RNA (2 µg) were mixed together and subjected to the MICROBEnrich procedure. Another mixed sample of total RNA was subjected to a mock MICROBEnrich procedure in which the capture oligonucleotide mix was left out of the reaction. RNA was precipitated and an equal volume of each sample was separated on an RNA Nano LabChip™ with the Agilent 2100 bioanalyzer.
Figure 1. RNA Before and After Enrichment with MICROBEnrich™ Kit. Rat liver total RNA (25 µg) and E. coli total RNA (2 µg) were mixed together and subjected to the MICROBEnrich procedure. Another mixed sample of total RNA was subjected to a mock MICROBEnrich procedure in which the capture oligonucleotide mix was left out of the reaction. RNA was precipitated and an equal volume of each sample was separated on an RNA Nano LabChip™ with the Agilent 2100 bioanalyzer.
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).
Figure 2. RNA Before and After Enrichment with MICROBEnrich™. Rat liver total RNA (25 µg) and E. coli total RNA (2 µg) were mixed together and subjected to the MICROBEnrich procedure with or without a further enrichment by MICROBExpress™. The total RNA lane was subjected to a mock MICROBEnrich procedure in which the capture oligo mix was left out of the reaction. RNA was precipitated and an equal fraction of each sample was analyzed by denaturing agarose gel electrophoresis.
Figure 2. RNA Before and After Enrichment with MICROBEnrich™. Rat liver total RNA (25 µg) and E. coli total RNA (2 µg) were mixed together and subjected to the MICROBEnrich procedure with or without a further enrichment by MICROBExpress™. The total RNA lane was subjected to a mock MICROBEnrich procedure in which the capture oligo mix was left out of the reaction. RNA was precipitated and an equal fraction of each sample was analyzed by denaturing agarose gel electrophoresis.
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.
Figure 3. Enrichment for Bacterial mRNA Results in More Sensitive Array Analysis. Replicate portions of Sigma/Genosys E. coli Panorama arrays were hybridized under identical conditions with 33P-labeled cDNA synthesized from (A) HeLa cell RNA (10 µg) mixed with E. coli RNA (800 ng) or (B) enriched E. coli mRNA that was purified from an identical mixture using MICROBEnrich™ and MICROBExpress™ Kits.
Figure 3. Enrichment for Bacterial mRNA Results in More Sensitive Array Analysis. Replicate portions of Sigma/Genosys E. coli Panorama arrays were hybridized under identical conditions with 33P-labeled cDNA synthesized from (A) HeLa cell RNA (10 µg) mixed with E. coli RNA (800 ng) or (B) enriched E. coli mRNA that was purified from an identical mixture using 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. A single place magnetic stand and Ambion's MICROBExpress Kit are available separately.
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