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Bacterial Whole Genome Array Analysis
Isolate and Enrich for Bacterial mRNA
Isolating Bacterial RNA for Microarray
Analysis
The rapid increase in the number of
bacterial genomes sequenced over the past several years has
opened up a new era in whole-genome expression analysis using
DNA microarray technology. In recent years, this tool has been
used to explore transcriptional profiles for a variety of bacteria
leading to a greater understanding of microbial metabolism
[1]. DNA microarray technology has also led to a better understanding
of microbial pathogenesis, physiology, epidemiology, ecology,
fermentation, and pathway engineering.
With the emerging importance of bacterial
microarray gene expression analysis, it is vital that bacterial
RNA isolated for array analysis be of the highest possible
quality. Key characteristics unique to bacteria known to significantly
impact RNA quality, and dramatically influence the quality
and validity of array analysis, include:
1. Growth phase at harvest
and harvest conditions
2. Rapid turnover of bacterial
mRNA
3. Bacterial mRNAs' lack
of a poly(A) tail, thus excluding the use of classical enrichment
methods (e.g. oligo(dT) enrichment)
The last of these factors was the basis
for developing Ambion's MICROBExpress™ Kit. MICROBExpress dramatically
increases the sensitivity of array analysis by removing >95%
of 16S and 23S rRNA from bacterial total RNA, leaving a highly
enriched population of bacterial mRNA. The enriched mRNA serves
as a superior template for synthesizing the labeled cDNA used
in whole genome array analysis.
Pre-Isolation Factors That Affect RNA Quality
There are several key factors to consider
before beginning any bacterial RNA isolation procedure. The
first is when to harvest the bacteria to
maximize yield and quality of intact RNA. The highest quality RNA is isolated
from cells in the logarithmic phase of growth. RNA isolated from bacteria in
the stationary phase typically exhibits increased degradation. For this reason,
it is highly recommended that RNA not be harvested from cells that have grown
into stationary phase unless a specific experiment requires such isolation.
A second factor to consider is the method
used to harvest the bacteria. It is known that bacterial mRNAs
exhibit a wide range of stabilities. For example, approximately
80% of all mRNAs in E. coli are known to have half-lives
of between 3 and 8 minutes [2]. For this reason, it is important
to collect cells in a manner that minimizes or eliminates the
impact of harvesting conditions on both gene expression profiles
and/or RNA quality. With this in mind, it is preferable to
process small volumes of bacterial cultures (1-2 ml) and to
use a brief centrifugation step (~1 min.) to pellet the cells.
The cell pellets can then be processed with one of the following
treatments:
• Immediate cell lysis
and RNA purification using RiboPure™-Bacteria
Kit
• Rapid freezing in liquid nitrogen (a freeze-thaw treatment may aid
lysis of some bacteria)
• Resuspension of cells in RNAlater® (see RNAlater Preserves Bacterial Gene Expression Profiles for Array Analysis).
These simple steps are of vital importance
for obtaining the highest quality RNA required in array analysis.
Isolating Bacterial Total RNA: RiboPure™-Bacteria
Traditional methods for isolation of
total RNA from bacteria require the use of hot phenol or include
other harsh techniques, some of which need specialized equipment.
For Gram-positive organisms, pretreatments with enzymes to
weaken the cell wall are often used prior to isolation of RNA.
However, it should be kept in mind that these enzymatic pretreatments
may alter gene expression patterns on a global scale and could
compromise array data.
The RiboPure-Bacteria RNA Isolation
Kit uses no enzymatic pretreatments and thus will not alter
gene expression profiles. RiboPure-Bacteria combines an efficient
glass bead and phenol-based RNAWIZ™-mediated
disruption step followed by a glass fiber filter-based RNA
purification step for high yields of exceptionally pure RNA.
Removing Contaminating Genomic DNA: TURBO
DNA-free™
Once the RNA is purified, it should
be treated with DNase to remove any contaminating genomic DNA.
This is especially important for array analysis and/or qRT-PCR.
Contaminating genomic DNA will be labeled during the cDNA synthesis/labeling
reaction and/or lead to non-specific RT-PCR products that could
interfere with quantitative results. TURBO
DNA-free is
ideal for this application (see The World's Best DNase).
Enriching for Bacterial mRNA: MICROBExpress™
For decades mRNA has been isolated from
eukaryotic sources using oligo(dT) selection. Bacterial mRNAs,
however, lack the relatively stable poly(A) tails found on
eukaryotic messages and thus cannot be enriched using affinity
selection with oligo(dT). Ambion's MICROBExpress Kit
offers enhanced signal sensitivity in gene expression analysis
across all array platforms tested (including glass, nylon and
GeneChip®) by its ability to remove >95%
of bacterial rRNA from total bacterial RNA preparations. The
depletion of rRNA leaves behind a highly enriched bacterial
mRNA population (See Figures 1 and Figure 2). Additionally,
MICROBExpress leads to an increase in the total number
of genes detected (i.e. increase in % present calls) and an
improvement in overall signal to noise on GeneChip E. coli Antisense
Genome Arrays (Figure 3).
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| Figure 1. Agilent® Bioanalyzer
Image of E. coli mRNA Purified Using MICROBExpress™. 16S
and 23S rRNAs were selectively removed from five different
10 µg samples of E. coli total RNA and the
remaining enriched mRNA fraction (~70 ng each) were run
in Lanes 2-6. A sixth 10 µg sample of the same
total RNA prep was subjected to a mock MICROBExpress procedure
without using the kit's rRNA capture oligo mix. Approximately
0.7 µg micrograms of the mock reaction was loaded
in Lane 7. As seen in the gel image the rRNA bands are
virtually undetectable in the mRNA obtained with the
MICROBExpress procedure versus the control lane. |
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Figure 2. Agilent® Bioanalyzer
Electropherograms of mRNA from Figure 1. As
seen in the (top) electropherogram, greater than 95%
of the bacterial 16S and 23S rRNA bands have been removed,
leading to a selective enrichment of mRNA species as
compared to the (bottom) mock MICROBExpress™ procedure.
(Relative fluorescence units have been rescaled between
the two electropherograms to allow a closer examination
of the MICROBExpress enriched RNA).
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Figure 3. 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.
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*Correlation coefficients were >0.99
for both replicate comparisons.
GeneChip® is a registered
trademark of Affymetrix, Inc.
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