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MICROBEnrich™ Kit
Your Data: Enriched Intracellular Bacterial RNA from Infected Bovine Cells
Dr. Adams’ laboratory (Texas A&M University)
primarily examines in vitro and in vivo host:pathogen interactions
with a focus on intracellular bacterial pathogens of cattle that
can be transmitted to humans. During the infection process, it
can be challenging to characterize the expression profile of
intracellular bacteria due to the difficulty of obtaining sufficient
quantities of intact, pathogen RNA that is free of host RNA for
downstream analyses. In an attempt to solve this problem, Dr.
Adams and colleagues developed a protocol that uses Ambion’s
MICROBEnrich™ Kit to preferentially deplete the
sample of eukaryotic RNA.
Carlos A Rossetti, Sara D Lawhon, L Garry Adams
Department of Veterinary Pathobiology, College of Veterinary
Medicine, Texas A&M University, College Station, TX, 77843-4467.
Phone: (979)845-9814 • Email: gadams@cvm.tamu.edu.
Brucella replicates within phagocytic cells
(monocytes and macrophages) of the host. Study of the infection
process, therefore, requires separation of bacterial RNA from
host eukaryotic RNA. As Dr. Adams’ work illustrates, the
MICROBEnrich™ Kit provides an ideal solution to this problem,
because it employs hybridization capture technology to remove
human, mouse, and rat mRNA and rRNA from complex host-bacterial
RNA populations, leaving behind enriched microbial total RNA.
Total eukaryote RNA (25 µg) from human
cervical carcinoma (HeLa S3, the positive control) or Madin-Darby
bovine kidney (MDBK) cell lines was mixed with 2 µg of
Brucella melitensis strain 16M RNA (ratio 12.5:1). The MICROBEnrich
Kit was then used to remove the eukaryotic RNA from the mixed
sample. The total RNA yield after treatment was 3.72 µg
(an 86.2% reduction) for the HeLa:Brucella mix and 4 µg
for the MDBK:Brucella mix (an 85% reduction). Evaluation of integrity
and composition of the RNA samples before and after treatment
confirmed that MICROBEnrich specifically depleted bovine RNA
from mixed host:pathogen samples. Data in Figure 1 indicate the
reduction of the eukaryotic 18S and 28S rRNA band, and enrichment
of the 16S and 23S prokaryotic rRNA bands in mixtures of Brucella RNA with both MDBK and HeLa S3 RNA (compare Lane 2 with Lane
1 and Lane 4 with Lane 3, respectively).
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| Figure 1. MICROBEnrich™ Kit Preferentially Depletes Eukaryotic RNA from Eukaryote:Prokaryote Mixed Samples. RNA from Madin-Darby bovine kidney (MDBK) or human cervical carcinoma HeLa S3 cells was extracted by TRI Reagent® (Ambion). Extraction of RNA from Brucella melitensis strain 16M was performed following a hot acid-phenol:chloroform procedure. Individual RNA samples were resuspended in DEPC-treated water containing 1% of RNase inhibitor protein. Contaminating genomic DNA was removed using the DNA-free™ Kit (Ambion), and samples were kept frozen at –80ºC until used. Total RNA was quantitated by A260 measurements (NanoDrop® ND-1000 Spectrophotometer), and the integrity of the RNA samples was confirmed using the Agilent® 2100 bioanalyzer. MDBK RNA (25 µg) and HeLa S3 RNA (25 µg; positive control) were each separately spiked with 2 µg of B. melitensis RNA, and the mixed samples were treated with the MICROBEnrich Kit (Ambion). Agarose gel electrophoresis image of 2 µg HeLa RNA:B. melitensis 16M RNA mix or MDBK RNA:B. melitensis 16M RNA mix pre-MICROBEnrich and post- MICROBEnrich treatment. |
MICROBEnrich Kit
The MICROBEnrich™ Kit removes >90%
of mammalian RNA from complex mixtures of host-bacterial samples
in less than 2 hours. The MICROBEnrich Kit comes with reagents
capable of depleting 500 µg of host cell RNA (20 rxns x
25 µg
RNA) and is scalable--a single reaction can process between 10
and 100 µg of total RNA. If desired, upon completion of
the MICROBEnrich Kit procedure, bacterial mRNA can be purified
from the total RNA through the seamless integration of Ambion’s
MICROBExpress™ Kit technology. This technology enables >95%
removal of prokaryotic 16S and 23S rRNAs.
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