what makes eukaryotic transcripts easier to isolate than bacterial transcripts?

• Remove >xc% of mammalian RNA from complex mixtures of host-bacterial samples
• Simple procedure takes less than 2 hours
• Works with any bacterial species
• Seamless integration with MICROBExpress™ Bacterial mRNA Purification Kit
• Enables microarray expression analysis with bacterial RNA isolated from host cells

Ambion's new MICROBEnrich™ (patent pending) is the just kit bachelor for bacterial RNA enrichment from mixed host-bacterial RNA populations. A robust yet simple process, MICROBEnrich depletes mammalian (host cell) RNA selectively, leaving behind highly enriched bacterial RNA.

Gene expression analysis following host-bacterial interactions is revolutionizing our agreement 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, considering no method has existed to isolate bacterial RNA abroad from host prison cell RNA, the respective analyses of bacterial transcriptomes has been impossible. Ambion now introduces a new applied science, MICROBEnrich, that enables whole genome microarray expression analyses of bacterial RNA afterwards 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 circuitous mixture of full RNAs is the starting material for the MICROBEnrich procedure. This host-bacterial cell total RNA mixture can be obtained by a diverseness of RNA isolation methods, ideally by Ambion's new RiboPure™-Bacteria Kit. Other methods including glass cobweb filter based methods (such as Ambion'southward RNAqueous™ Kits) and phenol based methods (such as Ambion'due south RNAwiz™ and the ToTALLY RNA™ Kit) are also appropriate. For microarray analysis and other sensitive applications, Ambion besides recommends a DNase treatment (such equally with DNAfree™ Treatment and Removal Reagents) to remove contaminating genomic Dna from the full RNA prior to the MICROBEnrich procedure.

Remove >xc% of Mammalian RNA from Circuitous Total RNA Mixtures

MICROBEnrich employs hybridization capture technology (patent pending) to remove human being, mouse, and rat RNA (both mRNA and rRNA) from complex host-bacterial RNA populations, leaving backside enriched microbial total RNA. In the beginning step of the MICROBEnrich procedure, host-bacterial total RNA is incubated with an optimized mixture of capture oligonucleotides that demark to the mammalian 18S and 28S rRNAs and polyadenylated RNAs. Adjacent, 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 ane shows an Agilent 2100 bioanalyzer electropherogram of a circuitous host-bacterial RNA population before and subsequently bacterial RNA purification with MICROBEnrich. The RNA profile indicates nearly complete removal (>95%) of the host 18S and 28S rRNAs.

Effigy one. RNA Before and Later Enrichment with MICROBEnrich™ Kit. Rat liver total RNA (25 µg) and Eastward. 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'south MICROBExpress engineering science. This technology enables >95% removal of prokaryotic 16S and 23S rRNAs (Figure 2).

Figure ii. RNA Earlier and Subsequently 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 past MICROBExpress™. The full 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

MICROB Enrich was optimized using E. coli RNA in a background of human being, mouse and rat RNA. However, the kit should perform well with RNA from whatsoever bacterial species. Although many mammalian host species may be uniform with MICROB Enrich, 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 meliorate templates than total RNA for downstream applications and therefore deliver dramatic increases in sensitivity when used in applications such as array analysis. Effigy 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 later on bacterial mRNA enrichment using the MICROBEnrich and MICROBExpress Kits.

Figure 3. Enrichment for Bacterial mRNA Results in More Sensitive Array Assay. Replicate portions of Sigma/Genosys E. coli Panorama arrays were hybridized nether identical weather condition 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 MICROB Enrich 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 unmarried reaction can process between ten 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 unmarried place magnetic stand and Ambion'southward MICROB Express Kit are bachelor separately.

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