- Dramatically increase the sensitivity of array analyses and other procedures
- Removes >95% of 16S and 23S rRNA from bacterial RNA
- Simple procedure takes less than 2 hours
- Works with many Gram positive and Gram negative bacteria
Rapid Purification of Bacterial mRNA is Now Possible
For decades mRNA has been isolated from eukaryotic sources using oligo(dT) selection. Bacteria, however, lack the relatively stable poly(A) tails found on eukaryotic messages. Thus, isolating mRNA from bacteria has been virtually impossible — until now. MICROBExpress employs a novel technology to remove >95% of the 16S and 23S rRNA from total RNA of E. coli and other bacterial species. The kit is suitable for mRNA purification from a broad spectrum of Gram positive and Gram negative bacteria. The mRNA isolated with MICROBExpress is a superior template for synthesizing labeled cDNA for array analysis, and is ideal for quantitative RT-PCR, Northern applications, and cDNA library construction.
Start With Any Total RNA Sample
Bacterial total RNA is the starting material for the MICROBExpress procedure. The RNA can be obtained by a variety of 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™). In the first step of the MICROBExpress procedure, total RNA is mixed with an optimized set of capture oligonucleotides that bind to the bacterial 16S and 23S rRNAs. Next, the rRNA hybrids are removed from the solution using derivatized magnetic microbeads. The mRNA remains in the supernatant and is recovered by ethanol precipitation.
The MICROBExpress procedure takes less than 2 hours. In addition to purifying bacterial mRNA that is virtually free of 16S and 23S rRNA, this method also delivers the benefits of quick processing, ease of handling, and scalability.
Highly Enriched Bacterial mRNA is Now a Reality
The MICROBExpress Kit removes the larger rRNAs that can make up as much as 80% or more of a bacterial total RNA sample. Analyses that previously could not be done for lack of mRNA are now possible.
Efficient removal of 16S and 23S rRNA from bacterial RNA dramatically increases sensitivity in downstream procedures such as Northern hybridization and array analysis. We demonstrated this by comparing array results from replicate Panorama™ arrays (Sigma Genosys) hybridized with labeled cDNA synthesized from either total bacterial RNA or from bacterial mRNA obtained using the MICROBExpress Kit. Eliminating rRNA from the RNA template prior to cDNA synthesis substantially increased the array hybridization signal.
Maximize mRNA Enrichment by Removing Small RNAs
MICROBExpress is designed to specifically remove 16S and 23S rRNAs; small RNAs such as tRNA and 5S rRNA are not removed. Using total RNA that lacks tRNA and 5S rRNA as the starting material for MICROBExpress produces the highest possible level of mRNA enrichment. Small RNAs can be removed during total RNA isolation by using Ambion's RiboPure™-Bacteria Kit. Alternatively, small RNAs can be removed from existing total RNA samples using Ambion's MEGAclear™ Kit.
What You Get
The MICROBExpress Bacterial mRNA Purification Kit comes with reagents for 20 mRNA purifications, each from 10 µg of total RNA. The kit also includes a control bacterial RNA sample and a detailed Instruction Manual with tips, troubleshooting advice, and references.
The MICROBExpress protocol requires the use of a magnetic stand, available separately.
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