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Technical Brief
High-Throughput Sample
Preparation from Whole Blood
for Gene Expression Analysis
Xingwang Fang,
Ph.D.
Xingwang Fang,* Kurt Evans, Roy C. Willis, Angela Burrell,
Quoc Hoang, Weiwei Xu, Mangkey Bounpheng, and Sharmili Moturi
Ambion, Inc., Austin, TX
Keywords:
blood,
RNA isolation,
globin mRNA
removal,
gene expression,
microarray
hole blood is an attractive sample source for
nucleic acid-based assays because it is readily
available, easily accessible, and rich in genetic information.
However, globin mRNA accounts for up to 70% of the
mRNA (by mass) in whole blood total RNA, resulting in
distortion of the RNA amplification and subsequently
causing decreased Present calls, decreased call
concordance, and increased signal variation in microarray
analysis. Therefore, for gene expression analysis, whole
blood is typically fractionated before total RNA isolation
to reduce globin mRNA content. We have developed
a high-throughput sample preparation technology that
streamlines workflows for (1) total RNA isolation from
whole blood (MagMAX-96 Blood RNA Isolation Kit),
(2) globin mRNA removal using a novel, nonenzymatic
technology (GLOBINcleardHuman Kit), and (3) mRNA
amplification and labeling for expression analysis
(MessageAmp II-96 aRNA Amplification Kit). Globin
mRNA removal eliminates the need for prefractionation
of whole blood, minimizing the potential for expression
profile changes during sample handling. Quantitative RTPCR showed that this method effectively removed up to
95% of the globin mRNA from the isolated RNA while
retaining normal levels of other mRNAs. The streamlined
sample preparation enables quick and accurate expression
W
*Correspondence: X. Fang, Ph.D., Senior Manager of Scientists,
Ambion, Inc., An Applied Biosystems Business, 2130 Woodward
Street, Austin, TX 78744-1832, USA; Phone: þ1.512.721.3701;
Fax: þ1.512.651.0201; E-mail: [email protected]
1535-5535/$32.00
Copyright
c
2006 by The Association for Laboratory Automation
doi:10.1016/j.jala.2006.10.001
analysis of relatively high numbers of blood samples. ( JALA
2006;11:381–6)
INTRODUCTION
In recent years, gene expression profiling, by means
of microarrays, has become a powerful, frequently
used tool for studying human diseases and their responses to drug therapies.1 The emergence of pharmacogenomic and personalized medicine in the
near future is projected to increase demand for use
of this technology. Whole blood is an ideal sample
source for gene expression profiling because it is
readily available, easily accessible, and rich in genetic information.2 A robust, automatable process
for preparing RNA from whole blood is needed to
enable high-volume studies.
Most useful genetic information in whole blood
resides primarily in the peripheral blood mononuclear cells, which make up approximately 0.1% of
the blood cellular fraction. Globin mRNA from
reticulocytes accounts for up to 70% (by mass) of
the total mRNA in the whole blood. When total
RNA from whole blood is amplified using the Eberwine linear amplification procedure,3 globin mRNA
product dominates the amplified RNA (aRNA).
This has been shown to decrease Present calls and
call concordance, and to increase signal variation.2,4e6 For this reason, blood is typically fractionated before total RNA isolation. However,
fractionation increases sample handling, which can
lead to RNA degradation and expression profile
changes. Also, blood fractionation is very difficult
to automate or to scale up for high-throughput
JALA December 2006
381
Technical Brief
processing because centrifugation is typically used in blood
fractionation procedures. For these reasons, an increasing
trend is to isolate total RNA from whole blood, followed
by globin mRNA reduction before amplification.
A typical approach for globin mRNA reduction is targeted degradation of globin mRNA by hybridization of total
RNA with oligonucleotides complementary to globin mRNA
and digestion of the mRNA/DNA hybrids with RNase H.5
This approach has been shown to significantly increase the
Present calls on GeneChip arrays with some change in call
concordance,5 probably due to degradation of RNA during
the treatment.
A novel, nonenzymatic globin mRNA reduction method
(GLOBINclear Kits) has been developed at Ambion.7 Biotin-labeled oligonucleotides complementary to multiple regions of both a- and b-globin mRNA are hybridized with
total RNA, and the biotin-labeled oligonucleotideeglobin
mRNA hybrids are then removed with magnetic streptavidin
beads. This approach efficiently removes both a- and bglobin mRNA while keeping other mRNAs intact.
We have developed a magnetic bead-based high-throughput total RNA isolation protocol from whole blood (MagMAX-96 Blood RNA Isolation Kit), which provides
a streamlined magnetic bead-based workflow for RNA isolation from whole blood. We describe here the integration of
three products in a semi-automated process for (1) sample
preparation from whole blood (MagMAX-96 Blood RNA
Isolation Kit), (2) globin mRNA removal (GLOBINcleard
Human Kit), and (3) RNA amplification, labeling, and purification (MessageAmp II-96 aRNA Amplification Kit), all in
a 96-well format. This streamlined workflow enables highthroughput expression analysis using whole blood samples.
MATERIALS
AND METHODS
Blood Collection
Whole blood (10 ml) was collected from two human donors in EDTA-containing Vacutainer tubes (BD). For each
donor sample, 300 mL aliquots were distributed to 24 wells
of a 96-well plate containing 600 mL lysis/binding Solution.
The blood was stored briefly (!10 min) in the collection
tubes at room temperature until addition to lysis plate,
described below.
RNA Isolation and Analysis
The MagMAX-96 Blood RNA Isolation Kit protocol was
modified to accommodate 300 mL whole blood samples, and
implemented on the KingFisher 96 Magnetic Particle Processor (Thermo Electron Corp.). The reagent volumes and layout of the KingFisher 96 Processor turntable are listed in
Figure 1. The lysis plate was prepared immediately before
starting the process to minimize RNA degradation. The
KingFisher 96 Processor completed the lysing, washing, genomic DNA digestion, and final RNA purification in !1 h.
RNA yield was quantified by A260 using a UVeVis spectrophotometer (NanoDrop Technologies), and its integrity
was examined by microfluidics capillary electrophoresis with
an RNA LabChip Kit on an Agilent 2100 bioanalyzer.
Quantitative RT-PCR (qRT-PCR) targeting PTGS2 and
TP53 was performed on a 7900HT Fast Real-Time PCR System using appropriate TaqMan Gene Expression Assays
(Applied Biosystems Inc.). An internal control transcript,
XenoRNA-01 Control RNA was used to monitor efficiency
of qRT-PCR reactions. XenoRNA-01 Control RNA is
Figure 1. MagMAX-96 Blood protocol scaled-up for the KingFisher 96 Processor. The layout of the KingFisher 96 Magnetic Particle Processor turntable is shown with the positions of plates containing appropriate MagMAX-96 Blood Kit reagents (Panel A). Upon completion of
a step, the turntable turns clockwise to move the plate containing the next reagent under the magnetic head. The reagent volumes, step
times, and plate sizes are also shown (Panel B). The step times include both mixing and bead collection.
382 JALA
December 2006
Technical Brief
1 kb long, and shows no sequence homology to any sequence
deposited at GenBank.
High-Throughput Globin mRNA Removal
Globin mRNA removal was performed with 0.3e1 mg
total RNA samples in triplicate using a GLOBINcleard
Human Kit (Ambion Inc.) following the standard protocol
from the instruction manual.7 The process was implemented
on a Biomek NX Laboratory Automation Workstation
(Beckman Coulter) equipped with an MJ Moto Alpha Thermocycler (MJ Research) and a 96-Well Magnetic-Ring Stand
(Ambion). The initial 50 C incubation steps were performed
in a 96-well PCR plate (Applied Biosystems), and all subsequent steps were performed in a 96-well U-bottom plate.
qRT-PCR was used to measure globin mRNA before and after the procedure, normalized to human RNA polymerase II
(hRPII) mRNA, to calculate the efficiency of globin mRNA
removal.
mRNA Amplification and Biotin Labeling
Globin-depleted total RNA was amplified and labeled using a MessageAmp II-96 aRNA Amplification Kit (Ambion), which is based on the Eberwine procedure.7 Briefly,
total RNA is reverse transcribed using an oligo(dT) primer
bearing a T7 promoter sequence. The cDNA undergoes second-strand synthesis and purification to yield a template for
in vitro transcription (amplification) with T7 RNA polymerase. Biotin-11-Uridine-52-triphosphate (Biotin-UTP) was
used in the transcription reaction to label the aRNA. Magnetic bead-based methods for both cDNA and aRNA purification8 were implemented on a Biomek Liquid Handler
(Beckman Coulter). The fully automated procedure has been
described elsewhere.9,10
Microarray Analysis
Microarray analysis was performed using GeneChip Human Genome U133 Plus 2.0 Arrays (Affymetrix), following
the standard protocol recommended by the manufacturer,
and expression profiles were analyzed with the manufacturer’s standard software.
RESULTS
AND DISCUSSION
Total RNA Isolation and Quality Examination
Endogenous ribonucleases are abundant in whole blood
and can quickly degrade RNA, especially upon cell lysis,11
if not effectively eliminated. The MagMAX-96 Blood RNA
Isolation Kit uses guanidine thiocyanate to lyse blood cells
and to denature nucleases12 while allowing RNA to bind to
magnetic beads. Genomic DNA is digested with TURBO
DNase under high salt conditions to minimize RNA degradation. For high-throughput preparation of total RNA,
magnetic bead-based technology offers the following advantages over glass fiber filter-based methods: (1) the beads can
be fully suspended in solution, enabling efficient binding,
washing and elution with reduced carryover of
inhibitors10,13; (2) the use of microsized beads permits elution
volumes as low as 20 mL, resulting in higher final RNA concentrations that are better suited for downstream processes;
and (3) it is more reliable for walk-away automation because
there is no potential for vacuum failure or clogging seen in
filter-based technology.
The MagMAX-96 Blood RNA Isolation Kit is designed
for isolation of total RNA from 50 mL whole blood in a regular 96-well plate, with a typical yield of w0.5 mg total RNA.
To obtain enough high-quality RNA for microarray analysis, we scaled up the MagMAX-96 protocol to 300 mL whole
blood. In addition, we implemented processing on a
KingFisher 96 Magnetic Particle Processor. As determined
by A260, 1.88 0.29 mg and 2.06 0.28 mg of total RNA were
isolated from 300 mL blood samples from Donor 1 and Donor 2, respectively (24 replicates each). Variation in RNA
yield between donors is common, mainly due to the difference in white blood cell counts among individuals. The consistency of the RNA yield from donor replicates indicates
that the RNA isolation process is robust.
To further illustrate the consistency of RNA yields using
the scaled-up MagMAX-96 Blood protocol, eight technical
replicates of RNA from each donor were randomly selected
and qRT-PCR was performed targeting two volatile blood
genes (PTGS2 and TP53; Fig. 2). While donor-to-donor variation was significant, as expected, variation among the technical replicates was very low (SD % 0.27Ct) showing that this
method is consistent and viable for isolating RNA for gene
expression analysis.
The standard GLOBINclear globin mRNA reduction
protocol is suitable for RNA samples up to 14 mL. Using
MagMAX-96 Blood elution volumes of 50 mL, R0.5 mg total
RNA could be used as input for the GLOBINclear protocol
without a need for vacuum concentration, allowing the two
procedures to be seamlessly linked.
The purity of total RNA isolated from whole blood is
a big concern, because whole blood is very rich in proteins,
which inhibit reverse transcription.14,15 To detect the presence of inhibitors in the RNA isolated using the scaled-up
MagMAX-96 Blood protocol, we added 0, 2, and 5 mL of
the extracted RNA to 15 mL qRT-PCR control reactions that
each contained 1000 molecules of a target XenoRNA-01
Control RNA. As shown in Figure 3, amplification of
XenoRNA-01 Control RNA was not inhibited by as much
as 5 mL extracted RNA (1/3 of the total qRT-PCR reaction
volume), demonstrating that inhibitors of qRT-PCR reactions were effectively excluded from the recovered RNA.
Because whole blood is rich in nucleases, total RNA isolated from whole blood is often degraded and not suitable
for microarray analysis. We examined the integrity of isolated RNA on an Agilent 2100 Bioanalyzer. Twelve samples
randomly selected from a 96-well plate had 28S:18S rRNA
ratios R1.1 (Fig. 4A and B); a ratio of O1.0 is typically considered indicative of RNA with a high degree of integrity for
total RNA isolated from whole blood. The RNA Integrity
Number (RIN) algorithm analyzes bioanalyzer information
JALA
December 2006 383
Technical Brief
Figure 2. Consistency of RNA isolated using the scaled-up MagMAX-96 Blood protocol. Eight randomly selected technical replicates (5 mL
each, 10% of the total preparation) of RNA from each donor were used in 15 mL qRT-PCR reactions targeting PTGS2 and TP53 with
TaqMan primers and probes.
from both rRNA bands, as well as information contained
outside the rRNA peaks (i.e., potential degradation products), to provide a more complete picture of RNA degradation states. The RIN values for these samples were R6.7
(Fig. 4B), generally considered to indicate a high degree of
integrity. The A260/A280 ratio for all 12 samples was O1.74
(Fig. 4B), indicating RNA free of contaminating proteins.
These measurements suggest that the scaled-up MagMAX96 Blood protocol delivered RNA sufficiently intact and pure
for high-quality microarray analysis.
were used in qRT-PCR reactions targeting a-globin, b-globin, and hRPII mRNA. Both a-globin and b-globin amplification showed significant shifts after processing by the
GLOBINclear globin reduction method; however hRPII remained unaffected by processing (data not shown). a-Globin
and b-globin mRNAs were reduced by O94% and O98%,
Effectiveness of Globin mRNA Reduction
Total RNA isolated from whole blood is very rich in a- and
b-globin mRNA. A good globin mRNA reduction protocol
should effectively remove both globin mRNAs while efficiently recovering the remaining RNA so that the expression
profile is maintained. To test these two parameters, three initial quantities of RNA isolated from whole blood were processed in triplicate in the high-throughput GLOBINclear
protocol. RNA recovery from the GLOBINclear processing
was measured by absorbance at 260 nm. As summarized in
Table 1, R80% of input RNA was recovered in all samples
after globin mRNA reduction.
To quantify the levels of a- and b-globin mRNA, 5 mL of
both the processed RNA and the original, unprocessed RNA
384 JALA
December 2006
Figure 3. Absence of qRT-PCR inhibitors in RNA isolation using
the scaled-up MagMAX-96 Blood protocol. One thousand copies
of XenoRNA-01 Control RNA were used in 15 mL qRT-PCR reactions targeting XenoRNA-01 Control RNA using TaqMan
primers and probes; 0, 2, or 5 mL of RNA isolated from six randomly selected samples were added to the reactions.
Technical Brief
Figure 4. Integrity of RNA isolated using the scaled-up MagMAX-96 Blood protocol. Twelve samples of total RNA were selected in a Vshaped pattern from an entire plate of 96 samples processed using the scaled-up MagMAX-96 Blood protocol. Each sample (1.2 mL) was
analyzed on an Agilent 2100 Bioanalyzer; results are rendered as a gel image (Panel A). The two predominant bands are 28S and 18S rRNA.
28S:18S rRNA ratios and RIN values were calculated using Agilent Technologies 2100 Expert Software (Panel B).
respectively, with various total RNA input (Table 2). Coefficient of Variance (CV) was %6% in all cases, except for
b-globin mRNA reduction in the 0.98 mg sample
(CV ¼ 14.5%).
The high efficiency of globin mRNA removal, coupled
with good RNA recovery, demonstrates that the MagMAX-96 Blood protocol followed by GLOBINclear processing provides an effective method for preparing RNA suitable
for successful mRNA amplification, labeling, and expression
profiling on a microarray.
The MessageAmp II-96 aRNA Amplification Kit was used
to amplify and label RNA on a Biomek Liquid Handler.8e10
Each aRNA (15 mg) was then hybridized to GeneChip Human Genome U133 Plus 2.0 Arrays. On average, samples
which had not been processed with the GLOBINclear Kit
had 17,052 Present calls, while 19,023 (w2000 or w12%
more) genes were called Present in samples which had undergone global mRNA reduction. This is consistent with what is
observed when the GLOBINclear protocol is carried out
manually in a single tube.16
mRNA Amplification and Expression Profiling on
Microarrays
CONCLUSION
The ultimate test of the quality of the isolated RNA and
the effectiveness of globin mRNA removal is the quality of
the microarray data after mRNA amplification/labeling.
Table 1. Recovery of RNA after globin mRNA reduction
Input
(total RNA, mg)
0.98
0.70
0.28
After globin
removal (mg)
0.78
0.59
0.27
% Recovered
79.6
84.3
96.4
Three input quantities of total RNA were processed in triplicate in the high-throughput
GLOBINcleardHuman protocol. RNA was quantified using a NanoDrop Spectrophotometer.
We have developed a fully streamlined sample preparation
workflow for whole blood samples for mRNA expression
profiling on a microarray. The use of magnetic bead-based
technology in each step enables high-throughput processing
and simplifies the robotic setup for automation. With this
semi-automated workflow, we were able to obtain high-quality total RNA from whole blood, effectively remove globin
mRNAs, and successfully amplify the mRNA, resulting in
high-quality microarray data, and 400 samples can be processed in an 8-h work day. With a more sophisticated robot,
the entire workflow could be fully automated, enabling
robust expression analysis with microarray technology in
a high-throughput format.
JALA
December 2006 385
Technical Brief
Table 2. Effectiveness of globin mRNA reduction
a-Globin
Input RNA (mg)
0.98
0.70
0.28
DDCta Processed/
unprocessed
5.0 0.5
4.3 0.1
4.2 0.2
b-Globin
% Remaining
3.3 1.2
5.1 0.3
5.5 0.7
DDCt Processed/
unprocessed
7.2 0.5
7.1 0.1
7.0 0.5
% Remaining
0.7 0.3
0.7 0.1
0.8 0.3
Both the processed RNA and the original, unprocessed RNA (5 mL each) as described in Table 1 were used in qRT-PCR reactions targeting a-globin, b-globin, and hRPII mRNA. a-Globin and b-globin
mRNA levels in processed (depleted) and unprocessed (total) samples were normalized to hRPII. aDDCt: [Ct(globin, depleted) Ct(hRPII, depleted)] [Ct(globin, total) Ct(hRPII, total)]; Ct: threshold cycle.
ACKNOWLEDGMENTS
Jose Santiago and Penn Whitley helped with the GLOBINclear protocol. We
thank Lisa Albright for editing the manuscript.
8. Willis, C.; Moturi, S.; Fang, X. High throughput aRNA amplification.
Ambion TechNotes 2005, 12(1). http://www.ambion.com/techlib/tn/
121/5.html (accessed August 21, 2006).
9. Cu, M.; Zhu, Z.; Willis, R. C. Automation of the MessageAmp II-96
aRNA Amplification System from Ambion using the Biomek 3000 Lab-
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