This Ambion® kit is for the purification of bacterial mRNA by removing rRNA from total RNA. Sufficient reagents for 20 mRNA purifications, each from 10 µg of total RNA are provided.
Features of the MICROBExpress™ Kit:
• 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
Isolating mRNA from bacteria has been virtually impossible, until now. The MICROBExpress™ Kit uses 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 the MICROBExpress™ Kit is a superior template for synthesizing labeled cDNA for array analysis, and is ideal for quantitative RT-PCR, northern applications, and cDNA library construction. Efficient removal of 16S and 23S rRNA from bacterial RNA dramatically increases sensitivity in downstream procedures (see Figure).
Start With Any Total RNA Sample
This host-bacterial cell total RNA mixture can be obtained by a variety of RNA isolation methods. The MICROBExpress™ Kit specifically removes rRNAs; small RNAs (i.e., tRNA and 5S rRNA) are not removed. Using total RNA that lacks small RNAs as the starting material for MICROBExpress™ Kit produces the highest possible level of mRNA enrichment (see Accessory Products). In the first step of the MICROBExpress™ Kit 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.
Accessory Products:
A magnetic stand is required for use this kit. They are available in several formats, including the Single Place Magnetic Stand (SKU #AM10026), 6 Tube Magnetic Stand (SKU #AM10055), and 96-well Magnetic Stands (SKU #AM10027 and #AM10050). The RiboPure™-Bacteria Kit (SKU #AM1925) is ideal for the purification of total RNA lacking small RNAs. Small RNAs can be removed from total RNA using the MEGAclear™ Kit (SKU #AM1908).
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![MICROBExpress&...](javascript: toggle("http://tools.invitrogen.com/content/sfs/gallery/high/f00343.jpg","MICROBExpress™ Analysis on the Agilent Bioanalyzer","16S and 23S rRNAs were selectively removed from a 10 µg E. coli total RNA sample using the MICROBExpress Kit (mRNA). 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. 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.)");)
![E coli mRNA Purif...](javascript: toggle("http://tools.invitrogen.com/content/sfs/gallery/high/f00344.jpg","E coli mRNA Purified with MICROBExpress™","16S and 23S rRNAs were selectively removed from a 10 µg E. coli total RNA sample using the MICROBExpress Kit (mRNA). 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 total RNA and mRNA samples were then analyzed by denaturing agarose gel electrophoresis. The rRNA is undetectable in the mRNA obtained with the MICROBExpress procedure.");)
![MICROBExpress&...](javascript: toggle("http://tools.invitrogen.com/content/sfs/gallery/high/f00345.jpg","MICROBExpress™ Enrichment of E coli RNA Improves Array Analysis","Replicate Panorama™ 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.");)
![Affymetrix&re...](javascript: toggle("http://tools.invitrogen.com/content/sfs/gallery/high/f00966.jpg","Affymetrix® GeneChip® E coli Antisense Genome Array Analysis","Illustrated above are Log Plots (Signal vs. Signal) for technical replicate GeneChips (i.e. same RNA, different probe labeling used for replicates). Panel A compares replicate arrays using 10 µg RiboPure™-Bacteria isolated E. coli total RNA with the standard Affymetrix labeling and hybridization protocol. Panel B compares replicate arrays utilizing the mRNA (0.69 µg) obtained with the MICROBExpress™ Kit from 10 µg of RiboPure-Bacteria isolated total RNA; this sample was labeled and hybridized using the same protocol as in Panel A. The total RNA (average between duplicate GeneChips) gave 73% Present Calls (genes above background) versus MICROBExpress isolated mRNA which gave 80% Present Calls, thus demonstrating an increase in sensitivity. In addition, the number of Concordant Calls for duplicate GeneChips increased with MICROBExpress mRNA (94%) compared with total RNA (92%). Concordant Calls are those genes called identically on both arrays (Present/Present, Marginal/Marginal or Absent/Absent), and is a direct measure of reproducibility.");)
