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GLOBINclear™-Human
Globin mRNA Removal Kit
Improved
Microarray Sensitivity using Whole Blood RNA Samples
Expression profiling with whole blood RNA samples has been difficult
in the past due to the heterogeneous cellular nature of blood
samples and the high concentration of globin mRNA present in
the RNA. This article describes how Ambion's new technology,
GLOBINclear-Human, significantly reduces the dilution effect
of globin mRNA on expression profiling using microarrays by removing
up to 95% of globin transcripts (>70% of the mRNA) from whole
blood RNA samples prior to amplification.
Challenges of Blood Sample Analysis
Expression profiling is widely used to study physiological,
pathogenic, and therapeutic responses of the transcriptome. For
clinical research, blood is the most commonly used tissue; but
blood samples present obstacles to gene expression analysis that
are not encountered with most other tissues. Blood is made up
of a heterogeneous population of erythrocytes, granulocytes,
and other peripheral blood mononuclear cells (PBMC). The complex
cellular nature of blood samples makes it difficult to detect
differential gene expression that occurs in only a subset of
cell types.
In addition, whole blood mRNA consists
of a relatively large proportion of globin mRNA transcripts.
Ambion scientists and others in the blood community, have estimated
that globin transcripts represent as much as 70% of the total
mRNA population. These “unwanted” globin
transcripts decrease the detection sensitivity of less abundant
mRNAs using microarray technology. Evidence of the dilution of
mRNA by highly abundant globin mRNA has been observed on Affymetrix® GeneChip® arrays,
where expression profiles from whole blood RNA show decreased
Present calls and increased variability among biological replicates,
relative to RNA from the white blood cell fraction of whole blood
(which does not contain globin mRNA).
The GLOBINclear™-Human Kit employs a novel, nonenzymatic technology
to remove >95% of the globin mRNA from whole blood total RNA.
The procedure is rapid and robust (Figure 1). First, globin mRNA
is removed from whole blood RNA using a novel hybridization capture
technology. Then, the remaining RNA is further purified with
a rapid magnetic bead-based clean-up procedure. The resulting
RNA is a superior template for RNA amplification or for synthesis
of labeled cDNA for array analysis. When used for expression
profiling, GLOBINclear-treated blood RNA delivers a significant
increase in sensitivity and a concomitant drop in variability,
relative to untreated whole blood RNA.
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Figure
1. The GLOBINclear™ Procedure.
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The GLOBINclear Procedure
To remove globin mRNA, 1–10 µg of total RNA from human
whole blood is mixed with a biotinylated Capture Oligo Mix in
hybridization buffer (Figure 1). The mixture is incubated for
15 minutes to allow the biotinylated oligonucleotides to hybridize
with the globin mRNA species. Streptavidin Magnetic Beads are
then added, and the mixture is incubated for 30 minutes. During
this incubation, streptavidin binds the biotinylated oligonucleotides,
thereby capturing the globin mRNA on the magnetic beads.
The Streptavidin Magnetic Beads are then pulled to the side
of the tube with a magnet and the RNA, depleted of the globin
mRNA, is transferred to a fresh tube. The RNA is further purified
using a rapid magnetic bead-based purification method. This simple
and fast (~30 minutes) purification consists of adding an RNA
Binding Bead suspension to the samples, and using magnetic capture
to wash and elute the GLOBINclear processed RNA.
RNA Recovery and
Amplification for Array Analysis
The GLOBINclear-Human procedure was primarily optimized for
RNA amplification using Ambion's MessageAmp II aRNA Amplification
Kit to synthesize RNA for microarray analysis. The goal was to
develop a process to improve global gene expression screening
of samples derived from human whole blood. Without GLOBINclear
processing, aRNA amplified from whole blood samples shows a very
distinct ~600 nt peak that represents amplified globin mRNA.
Ambion scientists found that total RNA from whole blood that
had been processed with GLOBINclear produced a very different
aRNA profile, in which the distinctive globin peak was absent
and the resulting aRNA produced a smooth curve (Figure 2). This
curve resulted from the successful removal of both alpha- and
beta- globin mRNA transcripts from the amplification reaction
and was more similar to the aRNA profiles obtained from
non-blood tissues.
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Figure 2. Biotinylated aRNA Amplified from Whole Blood RNA Processed with the GLOBINclear™-Human Kit. RNA from human whole blood (3 µg) was processed using the GLOBINclear-Human Kit. Then, either 1 µg of this GLOBINclear whole blood RNA or 1 µg of unprocessed whole blood RNA was amplified using the MessageAmp™ II-96 Kit to generate biotinylated aRNA. Equal mass amounts of aRNA were run on the Agilent® 2100 bioanalyzer. |
Figure 3 shows yield data for RNA treated with both GLOBINclear
and MessageAmp II RNA amplification.
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Figure
3. GLOBINclear™ RNA and aRNA Yields. Triplicate
GLOBINclear-Human reactions and MessageAmp™ II-96
RNA amplification reactions were performed with the indicated
input levels of whole blood total RNA or GLOBINclear
RNA, respectively. RNA and aRNA yield were determined
with a NanoDrop® spectrophotometer. Values in parentheses
represent 1 standard deviation. In the control reaction,
a whole blood RNA sample was amplified directly without
GLOBINclear processing.
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The GLOBINclear procedure typically provides RNA yields of 70–90%
of the starting material, depending primarily on the concentration
and integrity of the input RNA. Since GLOBINclear depletes samples
of >95% of their globin mRNA, (>70% of the mRNA present),
GLOBINclear-processed RNA yields less aRNA from MessageAmp II
amplification than an equivalent mass of unprocessed RNA. This
is because GLOBINclear-processed RNA actually contains far less
mRNA template for amplification in a given amount of total RNA
than unprocessed whole blood RNA.
To determine the amount of RNA to process with GLOBINclear,
consider the requirements of the downstream application. For
example, we recommend processing between 1 and 10 µg of
whole blood RNA with the GLOBINclear-Human Kit to prepare RNA
for amplification with MessageAmp II products for array analysis.
Note however, that more than 1 µg of input RNA may be required
if a different amplification kit is used.
Effective Globin mRNA Removal
Throughout development of the GLOBINclear-Human Kit, qRT-PCR
was used to assess depletion of alpha- and beta-globin mRNA.
Figure 4 shows representative qRT-PCR data for the GLOBINclear
RNA samples shown in Figure 3. TaqMan® probes for both alpha-
and beta- globin were used to assess the level of globin mRNA
before and after the GLOBINclear procedure. These data show that
both alpha- and beta-globin mRNA were reduced substantially by
GLOBINclear processing: alpha-globin mRNA was reduced between
40 and 60 fold (97.5–98.3% removal) and beta-globin mRNA was
reduced between 80 and 180 fold (98.8–99.4% removal). Notice
that there is an inverse relationship between globin mRNA depletion
and the amount of whole blood RNA processed. However, even with
this effect, globin mRNA depletion is still robust at the 7–10 µg
whole blood RNA input level.
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Figure 4. Alpha- and Beta-Globin mRNA Depletion Using the GLOBINclear™-Human Kit. Triplicate samples of the indicated amounts of whole blood total RNA were processed using the GLOBINclear-Human Kit. The RNA was then evaluated for globin mRNA reduction relative to untreated whole blood RNA using real-time qRT-PCR. Duplicate RT-PCRs were performed for each replicate. Fold change in globin mRNA levels were determined using the DDCt method. Notice that a 20-fold reduction in globin mRNA corresponds to 95% removal. |
Array Analysis with GLOBINclear-Human
To determine the effect of GLOBINclear processing on microarray
expression profiling, blood samples were drawn from 6 healthy
donors, and RNA was isolated with a modified PaxGene® (Qiagen)
protocol. RNA (3 µg from each donor) was processed with
the GLOBINclear Kit in triplicate. Figure 5 shows the amount
of remaining globin mRNA for each sample after depletion of globin
mRNA as described in Figure 4. In this experiment, alpha- and
beta-globin mRNA were depleted by well over 95% for all donor
samples
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Figure
5. Alpha- and Beta-Globin mRNA Depletion
of Human Blood Samples Assayed by qRT-PCR. Triplicate
GLOBINclear™ reactions (3 µg RNA input) were
performed on whole blood total RNA derived from 6 human
donors, and real-time qRT-PCR was used to monitor globin
mRNA depletion as in Figure 4. For all samples, globin
mRNA was depleted at least 50 fold (98.0% reduction)
relative to the untreated whole blood total RNA from
the corresponding donor.
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Next, 1 µg of each GLOBINclear-RNA sample was amplified
using the MessageAmp II-96 Kit to synthesize biotinylated aRNA.
Whole blood RNA (1 µg, unprocessed) was also amplified
in triplicate for each donor. Two of the three replicate amplification
reactions were randomly chosen and hybridized to Affymetrix GeneChip
U133 Plus 2.0 Arrays (6 donors x 2 replicates x 2 conditions = 24 arrays).
Increased Percent Present Calls: The sensitivity of
GLOBINclear RNA was evaluated by looking at several metrics,
including the percentage of genes called Present by Affymetrix
GeneChip Operating Software (GCOS). The percentage of genes called
Present increased dramatically (range, 9–17%; mean, 13.2%) as
a result of GLOBINclear processing of the RNA from all the blood
donors (Figure 6). An average of 7206 additional genes were called
present on U133 Plus 2.0 Arrays after GLOBINclear processing
(mean Present calls=21698 ± 606). This represents fully
50% more Present genes than were called for the unprocessed whole
blood RNA samples (mean Present calls=14492 ± 1347). Interestingly,
the variability in sensitivity (Percent Present call standard
deviation) seen within and between donors was significantly reduced
after GLOBINclear processing. The exact reasons for this decreased
variability are unknown; however, the most likely cause is the
low signal-to-noise ratio generally associated with untreated
whole blood RNA. The variability does not seem to be caused by
differences in the levels of globin mRNA concentration between
donors (data not shown).
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Figure
6. Percent
Present Call Data for 6 Blood Donors on Affymetrix® GeneChip® U133
Plus 2.0 Arrays. Two of the three replicate
GLOBINclear™- processed RNA samples (1 µg)
for each donor shown in Figure 5 were amplified using
the MessageAmp™ II-96 Kit to synthesize
biotinylated aRNA. Untreated whole blood RNA from each
donor was amplified in parallel. The biotinylated aRNA
was then hybridized to duplicate microarrays, and the
number of Present calls were determined using Affymetrix
GeneChip operating software (GCOS) using the default
settings, with scaling to all probe sets and a target
signal value of 500. The error bars represent 1 standard
deviation. RNA samples processed with the GLOBINclear-Human
Kit resulted in a clear and consistent increase in
genes called present (50% increase).
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Decreased 3'/5' Ratio: Another instance of increased
variability associated with untreated whole blood RNA seen during
the course of this study was 3'/5' ratios in array analysis.
aRNA from samples processed with the GLOBINclear-Human Kit showed
a significant decrease in 3'/5' ratios of the housekeeping genes,
GAPDH and actin, compared to aRNA from unprocessed samples (Figure
7). 3'/5' ratios decreased from an average of 4.43 for GAPDH
and 8.03 for actin in whole blood samples, to 1.65 for GAPDH
and 1.64 for actin in GLOBINclear-processed samples, respectively.
In the last row of the table, results were recalculated after
removing the single outlier array from Donor D, and 3'/5' ratios,
and the standard deviations decreased even further. The high
3'/5' ratios seen with untreated whole blood samples are most
likely due to decreased sensitivity.
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Figure
7. Affymetrix® GeneChip® 3'/5' Ratios
for Housekeeping Genes. Average GAPDH and Actin
3'/5'signal ratios are shown for GeneChip arrays hybridized
with aRNA amplified from GLOBINclear-processed RNA or
untreated whole blood RNA. Average values are derived
from all 12 arrays (6 donors x duplicate technical replicates).
The last row shows average ratios after removing results
from the donor D outlier array. Affymetrix recommends
that GAPDH and actin ratios be below 2.0 and 4.0, respectively.
Numbers in parenthesis represent 1 standard deviation.
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Reproducibility: The GLOBINclear method was further
assessed for reproducibility by examining both the technical
replicate concordance values and the correlation coefficients
between replicates. Total concordant calls [P-P (Present), M-M
(Marginal), A-A (Absent)], and both technical and biological
correlations, were demonstrably higher for GLOBINclear-processed
samples, compared to untreated whole blood samples (Figure 8).
Here, as demonstrated in Figure 7, if the Donor D outlier is
removed from consideration, the average concordance and average
technical replicate correlation is further increased to 90.2%
and 0.995, respectively. Also shown in Figure 8 are correlation
coefficients for samples from donors B–F compared with a baseline
sample from Donor A. As with the technical replicate comparisons,
processing with GLOBINclear also reduces the variation seen among
biological replicates.
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Figure
8. Correlation and Percent Concordance
for GLOBINclear™ RNA and Untreated Whole Blood
RNA. Pearson’s correlation coefficient
(r) and total percent concordance are presented for biological
and technical replicates. All correlation coefficients
were derived from signal values estimated using Robust
Multichip Average (RMA, www.bioconductor.org). Concordance
is the number of concordant calls [P-P (Present), M-M
(Marginal), or A-A (Absent)] divided by the total number
of calls (or genes = 54,675). Present/Marginal/Absent
calls were determined with Affymetrix® GeneChip Operating
Software (GCOS).
Note 1: RMA signal values were averaged
for donor technical replicates and correlation coefficients
were computed for all donors vs. Donor A (baseline).
Note 2: Concordance and correlation coefficients
for technical replicates were computed for each donor.
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Conclusion
The GLOBINclear-Human Kit utilizes a biotin/streptavidin magnetic
bead capture method that removes more than 95% of unwanted alpha-
and beta-globin mRNA transcripts from human whole blood RNA.
GLOBINclear-processed whole blood RNA provides increased sensitivity
and decreased variability when used for microarray expression
analysis. The method is simple, highly reproducible, and takes
less than 1.5 hours to complete.
The kit comes with enough reagents to process 20 globin mRNA
depletion reactions. Whole blood RNA isolation kits and magnetic
stands are sold separately.
Scientific Contributors
Penn Whitley, Sharmili Moturi, Jose Santiago, Charles Johnson,
Robert Setterquist • Ambion, Inc.
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