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.
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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.
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Compatible Bacteria
The MICROBExpress Kit has been verified to function with E. coli, B. subitlis, N. meningitidis, and S. aureus. Ambion has searched the sequenced genomes and incomplete genomes on the NCBI's Microbial Genomes web page to determine if MICROBExpress is compatible with additional organisms listed there.
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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.)
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 Panoramaþ 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 Panoramaþ 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.
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 Panoramaþ 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 Panoramaþ 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..
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.
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