Tips
for Optimizing Bacterial Array Analysis
Isolating Bacterial RNA
from Eukaryotic
Host Cells
Challenges for Studying Bacterial Host Interactions
Gene expression studies of bacterial pathogens following host cell interaction
have been especially challenging (1, 2). Namely purifying
adequate amounts of high quality RNA for bacterial transcriptome analysis has
proven to be difficult. Harvesting bacteria and purifying
bacterial RNA from infected tissues or in vitro cell cultures without altering
gene expression is the first problematic step. In addition, bacterial cell
numbers in diseased tissues or organs are frequently small in comparison to
the numbers of host cells present. Even when host cells are infected in vitro,
the numbers of bacteria that adhere to or
invade cells is often low. Therefore, purifying total RNA from a mixture of
eukaryotic cells and bacteria most often results in a vast excess of eukaryotic
cellular RNA and very little bacterial RNA (Figure 1). Frequently too little
bacterial RNA is present for generating adequate signals on microrarrays.
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| Figure 1. Optimal
RNA Yields from Mixtures of Bacteria and Eukaryotic Cells. 1 E.coli and
HeLa cell average optimal RNA yields. |
Start By Preserving RNA Expression Profiles
For host-pathogen interaction studies,
using Ambion's RNAlater™ to "freeze" RNA
expression profiles and preserve RNA integrity is an essential first step prior
to harvesting cells (Figure 2). Infected tissues or organs should be immersed
in RNAlater immediately upon removal from the host organism. RNAlater will
penetrate bacterial and host cell membranes and rapidly inactivate endogenous
ribonucleases and other cellular enzymes so that the RNA is left intact and
its expression pattern unaltered. With cultured cells, it is best to first
remove culture medium then add RNAlater to the culture flask or plate
so that all cells are completely covered. Allow approximately 10 minutes for
RNAlater penetration before harvesting cells for RNA isolation. For
non-adherent eukaryotic cells, collect cells by centrifugation, then thoroughly
resuspend them in RNAlater.
For bacteria that have adhered to host cells but are not internalized, it may
be desirable to wash cells prior to the addition of RNAlater to remove
bacteria that are not adhering tightly.
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| Figure 2. Recommended
Procedures for Purifying Bacterial mRNAs for Whole Genome
Expression Analysis Following Bacteria-host Cell Interactions. |
Isolating Bacterial RNA
From Host-Pathogen Mixtures
After freezing expression profiles and harvesting
cells, RNA is purified from the host-pathogen cell mixture. One approach
is to isolate RNA from the entire
host-pathogen mixture using standard lysis buffers. However, bacteria present
in the mixture may not be lysed without harsher methods for cell disruption,
such as sonication (3). A more effective approach for increasing yields of
bacterial RNA from host-pathogen mixtures is to selectively lyse the host
cells, remove host RNA and cellular debris, and then isolate RNA from the
bacteria using cell disruption methods appropriate for bacteria, such as
Ambion's RiboPure™ Bacteria Kit.
One example of such an approach was reported by Grifantini et al. (4) who
used saponin-mediated lysis (1% w/v) of epithelial
cells prior to purifying RNA from adherent meningococci. Saponin-mediated
lysis has also been used to release intraerythrocytic Plasmodium falciparum (5)
and for detection of intracellular pathogenic bacteria in cerebrospinal fluid
(6). Saponins can also be used in the presence of RNAlater,
which preserves RNA expression profiles during host-cell lysis. Because not
all eukaryotic cell types are susceptible to saponin-mediated lysis, this procedure
should be evaluated with the host cell line of interest prior to implementation
(see the Sidebar, Saponin Mediated Lysis of Eukaryotic Cells Harboring Bacterial
Pathogens, for a brief protocol).
Following selective lysis of host cells, bacteria
can be harvested and RNA purified with Ambion's RiboPure Bacteria
Kit. RiboPure
Bacteria is the only commercially available kit containing all
reagents necessary for phenol-extraction and glass fiber filter
purification of bacterial RNA. It is also compatible with RNAlater.
RNA purified with RiboPure Bacteria is of extremely high quality
and well suited for genome expression analysis with DNA arrays.
Enriching for Bacterial mRNA
For optimal cDNA synthesis and labeling
prior to array analysis, it is useful to remove contaminating
eukaryotic RNA from any unlysed host cells remaining
with the bacterial cells. Ambion's MICROBEnrich™ Kit (patent
pending) was developed specifically to deplete eukaryotic RNA from mixtures
of bacterial and host cell RNA. The MICROBEnrich Kit can be used with
large quantities of eukaryotic RNA, even when small amounts of bacterial
RNA are present in the mixture, such as when saponin-mediated lysis of host
cells is not an option.
The MICROBEnrich Kit employs a novel technology
to selectively remove >90% of human, mouse, or rat total RNA from a mixed
population of mammalian and prokaryotic RNA. The remaining bacterial RNA can
then be used in downstream applications such as array analysis. The MICROBEnrich reaction
is scalable (up to 100 µg). The kit can be used to remove a total of 500 µg
of mammalian RNA from a mixed population of mammalian/bacterial RNA. Typically,
the MICROBEnrich Kit is used for 20 reactions, each consisting of mixed
RNA samples containing up to 25 µg of mouse, rat, or human RNA. For further
enrichment of bacterial mRNA, 16S and 23S bacterial rRNA can be removed from
the bacterial total RNA using Ambion's MICROBExpress™ Kit. The
MICROBEnrich procedure can be seamlessly integrated with Ambion's MICROBExpress Kit
for elimination of bacterial large subunit rRNAs by further enrichment of bacterial
mRNA.
References
1. Sassetti C, Rubin EJ. (2002)
Genome analyses of microbial virulence. Curr Opin Microbiol 5: 27-32.
2. Cummings CA, Rehlman DA. (2000)
Using DNA microarrays to study host-microbe interactions. Emerg
Inf Dis 6: 513-25.
3. Staudinger BJ, Oberdoerster
MA, Lewis PJ, Rosen H. (2002) mRNA expression profiles for Escherichia
coli ingested
by normal and phagocyte oxidase-deficient human neutrophils.
J Clin Invest 110(8): 1151-63.
4. Grifantini R, Bartolini E,
Muzzi A, Draghi M, Frigimelica E, Berger J, Ratti G, Petracca
R, Galli G, Agnusdei M, Giuliani
MM, Santini L, Brunelli B, Tettelin H, Rappuoli R, Randazzo F,
Grandi G. (2002) Previously unrecognized vaccine candidates against
groupB meningococcus identified by DNA microarrays. Nature
Biotechnol 20: 914-21.
5. Hsiao LL, Howard RJ, Aikawa
M, Taraschi TF. (1991) Modification of host cell membrane lipid
composition by the intra-erythrocytic
human malaria parasite Plasmodium falciparum. Biochem
J 274: 121-32.
6. Gould FK, Freeman R, Law D,
Moriarty T. (1988) Lysis in detection of intracellular organisms. Lancet 2: 461.
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