For the separation and isolation of bacterial RNA from mixed samples. The Ambion® kit includes sufficient reagents for depleting 500 µg of host cell RNA (20 reactions x 25 µg RNA) from a complex host-bacterial RNA mixture. 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. The MICROBEnrich™ Kit enables whole genome microarray expression analyses of bacterial RNA after host-bacterial interactions.
Start With Any Total RNA Sample
For microarray analysis and other sensitive applications, a DNase treatment is recommended to remove contaminating genomic DNA from the total RNA prior to the MICROBEnrich™ Kit procedure.
Remove >90% of Mammalian RNA from Complex Total RNA Mixtures
The MICROBEnrich™ Kituses 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 procedure, host-bacterial total RNA is incubated with a mixture of capture oligonucleotides that bind 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. An Agilent 2100 bioanalyzer electropherogram (see Figure) shows nearly complete removal (>95%) of the host 18S and 28S rRNAs from a mixed sample. Bacterial mRNAs purified with the MICROBEnrich™ Kit are better templates than total RNA for downstream applications and therefore deliver dramatic increases in sensitivity when used in applications such as array analysis (see Figure).
The MICROBEnrich™ KitWill Work With Any Bacterial Species
The MICROBEnrich™ Kit 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 the MICROBEnrich™ Kit, only human, mouse, and rat rRNA sequences were used for the design and optimization of the capture oligonucleotides.
A magnetic stand is required for use this kit. They are available in several formats, including the Single Place Magnetic Stand (SKU #AM10026), 6 Tube Magnetic Stand (SKU #AM10055), and 96-well Magnetic Stands (SKU #AM10027 and #AM10050). The RiboPure™-Bacteria Kit (SKU #AM1925) is ideal for the purification of host-bacterial cell total RNA. The TURBO™ DNA-free™ or DNA-free™ DNase Treatment and Removal Reagents (SKU #AM1907 and AM1906, respectively) are recommended for microarray analysis and other applications where the removal of contaminating genomic DNA is needed.After the MICROBEnrich™ procedure, bacterial mRNA can be purified from the total RNA using the MICROBExpress™ Bacterial mRNA Enrichment Kit (SKU #AM1905). This technology enables >95% removal of prokaryotic 16S and 23S rRNAs.