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. |
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.
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.
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
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
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..