FINALLY! Purify Bacterial mRNA

Ambion introduces MICROBExpress™ (US patent pending), the only bacterial mRNA purification kit available. Using a novel strategy and a rapid, simple procedure, MICROBExpress removes virtually all of the large ribosomal RNA from bacterial total RNA samples.

• Dramatically increase the sensitivity of array analyses and other procedures
• Remove >95% of 16S and 23S rRNA from E. coli total 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 (Figures 1 and 2). The kit is suitable for mRNA purification from a broad spectrum of Gram positive and Gram negative bacteria (see sidebar, "Compatible Bacteria"). The mRNA isolated with MICROBExpress is a superior template for synthesizing labeled cDNA for array analysis (Figure 3), 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 Ambion's RNAwiz™ and 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.

Figure 1. MICROBExpress™ Analysis on the Agilent 2100 Bioanalyzer. 16S and 23S rRNAs were selectively removed from a 10 µg E. coli total RNA sample using the MICROBExpress Kit. A second 10 µg sample of the same total RNA prep was subjected to a mock MICROBExpress procedure without the Capture Oligo Mix (total RNA). The electropherograms above were generated by running each RNA sample on an Agilent 2100 bioanalyzer using the RNA LabChip (see the Agilent web site for more information). Aligning the traces reveals >95% removal of the 16S and 23S rRNA peaks in the mRNA purified with the MICROBExpress Kit. (Note that the peak corresponding to 5S rRNA and tRNAs is not affected by the MICROBExpress procedure.)

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.

Figure 2. E. coli mRNA Purified with MICROBExpress™. The total RNA and mRNA samples described in Figure 1 were analyzed by denaturing agarose gel electrophoresis. 23S and 16S rRNA are undetectable in the mRNA obtained with the MICROBExpress procedure.

Highly Enriched Bacterial mRNA Makes A Better Template

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. Figure 1 shows an Agilent 2100 bioanalyzer electropherogram of a bacterial mRNA sample before and after mRNA purification with MICROBExpress. The reduction of the 16S and 23S rRNA peaks in the enriched sample indicates nearly complete removal of these RNAs from the sample. In Figure 2, E. coli RNA before and after removal of rRNA is visualized on a denaturing agarose gel. The data show >95% removal of the 16S and 23S rRNA.

Efficient removal of 16S and 23S rRNA from bacterial RNA dramatically increases sensitivity in downstream procedures such as Northern hybridization and array analysis. Figure 3 demonstrates this by comparing array results from replicate Panoramaž arrays (Sigma Genosys) hybridized with labeled cDNA synthesized from either bacterial total 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.

Figure 3. MICROBExpress™ Enrichment of E. coli RNA Improves Array Analysis. Replicate Panoramaž E. coli arrays (Sigma-Genosys) were hybridized with 32P labeled cDNA generated by reverse transcription of E. coli RNA using random hexamers. The bottom array was hybridized with cDNA made from 8 µg of total RNA. A second 8 µg aliquot of the same RNA was enriched for mRNA using the MICROBExpress Kit and used to prepare labeled cDNA for hybridization to the top array. After removal of unincorporated radiolabel, the entire cDNA labeling reactions were used for the array hybridization.

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 one of Ambion's RNAqueous® Kits. Alternatively, small RNAs can be removed from existing total RNA samples using Ambion's MEGAclear™ Kit (Figure 4).

Figure 4. Efficient Removal of 5S rRNA and tRNA with MEGAclear™. Electropherogram of a total RNA preparation before and after purification with MEGAclear illustrates removal of the low molecular weight 5S rRNA and tRNA by MEGAclear procedure.

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. A single place magnetic stand is available separately for use with MICROBExpress.

Related Articles

Organisms Compatible with the MICROBExpress™ Bacterial mRNA Isolation Kit

MICROBExpress™ Instruction Manual