|
Improving RNA Amplification
Performance of New MessageAmp™ II
on
High Density Oligonucleotide Microarrays
• New
ArrayScript™, an M-MLV RT engineered to maximize yields
of full-length cDNA
• Amplify
as little as 100 ng of input RNA in a single round for GeneChip® analysis
• cRNA
synthesis powered by MEGAscript®, the best technology for
in vitro transcription
• High
labeling efficiency yielding consistently high percent Present
calls, and low 3'/5' ratios in
GeneChip analysis
RNA amplification is often required for producing
sufficient amounts of labeled target nucleic acid for detection
by array hybridization. The original T7 RNA polymerase linear
amplification method described and patented by Van Gelder and
Eberwine [1-3] is the basis for sample preparation for most commercial
microarray platforms, including the high quality Affymetrix GeneChip®.
At Ambion we recognize that there is more to improving RNA amplification
than simply increasing yields of labeled cRNA. We therefore,
set out to ensure that any observed improvements in amplification
coincide with accepted standards for high quality expression
profiling.
RNA Amplification Enhancements
Ambion is recognized as one of
the key research companies for development of comprehensive solutions
for cRNA (aRNA) amplification. Ambion’s MessageAmp™ aRNA
Amplification Kit was the first commercially available Eberwine-based
aRNA amplification kit. The MessageAmp II cRNA Amplification
Kit incorporates extensive improvements to the original MessageAmp
procedure. In developing MessageAmp II, Ambion’s
scientists focused on the reverse transcription and second strand cDNA synthesis
steps, both of which are critical for generating high yields of full-length
labeled cRNA. This led to the development of ArrayScript™, an engineered
M-MLV reverse transcriptase, and to the optimization of the second strand cDNA
synthesis reaction to synergize with the improvements gained with ArrayScript.
One of the principal benefits conferred by ArrayScript and
the optimized second-strand synthesis step is that MessageAmp II will generate
sufficient labeled cRNA in a single round of amplification for array analysis
from as little as 100 ng of total RNA (Figure 1). Other commercially available
products for cRNA amplification usually demand two rounds of amplification
at this level of input.
|
|
|
Figure 1. High
Density Oligonucleotide Microarray Hybridized with MessageAmp™
II cRNA. Shown
is a portion of a GeneChip® array image used in this
report. All RNA samples were amplified and labeled with
biotin-16-UTP/-11-CTP (PerkinElmer Life Sciences), with
the MessageAmp II Kit. Biotin labeled cRNA was fragmented,
then hybridized and stained using GeneChip Human Genome
Focus Arrays following the GeneChip Expression Analysis
technical manual.
|
Together, the RT and second strand cDNA reactions
result in an increase in the conversion of mRNA into longer double-stranded
cDNA templates. This increase allows both shortened in vitro
transcription (IVT) reaction incubation times and more reliable
access to expression information from total RNA samples less
than 1 µg. The increase also translates through to two
rounds of amplification, allowing more robust amplification of
total RNA from 10 ng to as low as 100 pg.
Expectations for Yields
Factors such as tissue source, mRNA content
(0.1-3%), starting amount of total RNA, and the quality of RNA
used, will all affect the yield of cRNA. Collectively these factors
can lead to up to 4-fold variation in yields. Figure 2 provides
typical yields of cRNA amplified from six different sources using
the MessageAmp II aRNA Amplification Kit. RNA inputs ranging
from 50-3000 ng were tested from each source. From Figure 2
one can calculate the level of amplification obtained from
different tissues at different inputs. The figure is also a
useful benchmark for understanding the amount of cRNA produced
both from different total RNA inputs and sources.
|
|
|
Figure 2. MessageAmp™
II aRNA Amplification Yields. Yields
of cRNA (µg) amplified using the MessageAmp II
aRNA Amplification Kit from six different RNA sources
using total RNA inputs of 50-3000 ng. Yields shown are
the average of duplicate reactions. The in vitro transcription
reaction incubation time was 4 hours for all samples.
Purified cRNA concentrations were measured using a NanoDrop®
spectrophotometer.
*Universal Human Reference RNA (Stratagene)
|
One or Two Round Amplification?
Based on specific experimental
constraints, researchers must decide whether one or two rounds
of amplification are appropriate. Some microarray studies demand
a high level of amplification (106 fold or greater), which can
be achieved by two successive rounds of MessageAmp II reactions.
Determining the lowest level of total RNA input that generates
enough cRNA for array hybridization will depend on the amount
of cRNA required for a particular microarray platform, and whether
extra cRNA is desired for replicates, follow up validation with
RT-PCR, or for archiving samples.
For consistency within an expression
experiment, it is important to decide whether one or two rounds
of amplification will be used throughout the study. For example,
yields of total RNA may vary within a collection of samples – some samples may
yield 1 µg of total RNA such that only one round of amplification
is needed, whereas others may only yield 50 ng, requiring two
rounds of amplification. Figure 3 may aid in this decision process
for amplifying samples below 100 ng. For example, below 100 ng
input RNA, the cRNA yields after one round of amplification may
not be sufficient for most arrays.
|
|
|
Figure 3. cRNA
Yields From Two Rounds of MessageAmp™ II Amplification. 100,
10, and 0.1 ng of total RNA from HeLa S3 cells (HeLa)
and human inferior temporal cortex (brain) were amplified.
Yields (µg) using the MessageAmp II aRNA Amplification
Kit are recorded for after each round of amplification.
The starting cRNA input for the second round was 2 µg
(if available), or the entire amount obtained from the
first round.
|
Obtaining sufficient cRNA for a microarray
hybridization is not only dependent on the source of RNA being
amplified but also on two reaction variables: the amount of
input total RNA, and the length of the IVT incubation. The
yield of cRNA increases with increasing RNA input and IVT time.
As a rule, most samples between 100-1000 ng, incubated overnight
(14 hr IVT), will produce sufficient cRNA for any microarray
platform (at least 10 µg).
However when we investigated a range of IVT incubation times
(from 4-14 hr), we found 4 hour IVT incubations generate sufficient
cRNA from most total RNA inputs greater than 200 ng (Figure 4).
A 4 hour IVT can be sufficient for 100 ng samples, and these
conditions were used in the microarray studies presented below.
(Note that mRNA poor samples will require longer IVT incubations
(6-14 hr).
|
|
|
Figure 4. Plot
of IVT Incubation Time vs. Yield. Total
RNA
(200 ng) from human inferior temporal cortex (brain)
was amplified and incubated with in vitro transcription
(IVT) reaction times of 4, 6, and 14 hours following
the MessageAmp™ II protocol. The average yield
of purified cRNA from duplicate amplification reactions was plotted versus
the IVT incubation time. The 0,0 data point was added
to extend the line to predict yields with IVT incubation times under
4 hours.
|
While we recommend a minimum of 4 hours for
the IVT incubation, yields obtained from shorter time periods
(2 or 3 hr) can be tested and should be adequate for input amounts
above 500 ng.
Expression Analysis
Three approaches were used to define the
reproducibility and consistency of the MessageAmp II protocol.
First, the relative consistency of the MessageAmp II protocol
was measured by comparing replicate arrays, where amplified samples
originated from the same RNA source, either inferior temporal
cortex from human brain (brain) or HeLa S3 cells (HeLa). These
cRNA samples were hybridized to GeneChip Human Genome Focus Arrays
following the Affymetrix guidelines. For a reference series we
used
the Affymetrix GeneChip 3'-Amplification Reagents One-Cycle cDNA Synthesis
Kit and IVT Labeling Reagents (from here on referred to as the Affymetrix protocol)
with duplicate 1000 ng reactions of the same RNA. The Affymetrix quality assessment
guidelines were met for all arrays in this study. This included 3'/5' ratios
(under 2.0), percent Present calls (50-53%), scaling factors, average background,
as well as visual inspection of images. This assessment indicated that all
arrays were of high quality.
MessageAmp II Reproducibility. Signal
values for each probe set were converted from .CEL files into
a summary of expression values measured for each of the 8793
probe sets (genes). Expression values were estimated using
Robust Multi-array Average (RMA) [4-6] as implemented in BioConductor
(www.bioconductor.org). The correlation established by this
comparison shows that amplification using these protocols with
1000 ng of input total RNA is highly reproducible (Figure 5).
|
|
|
Figure 5. (A)
Replicate Analysis of Two Amplification Protocols. Normalized
signals are plotted from replicate amplifications. Both
MessageAmp™ II (top) and Affymetrix standard
protocol (bottom) were performed with the identical total
RNA (1 µg) and equivalent amounts were hybridized
on matched lot Human Genome Focus arrays. All IVT reactions
were performed for 4 hours. Correlation values are included
and indicate a measure of reproducibility with both methods.(B)
Correlation and Standard Deviation of Differences for
RNA Inputs and IVT Incubation Times Compared to Standard. Two
sources of total RNA brain and HeLa S3 cells, were used
for amplification, and 4, 6, and 14 hr IVT reaction times
were tested. For comparison the same information is provided
for replicates of the Affymetrix protocol.
|
Differential Gene Expression. We also addressed differential
gene expression between two different tissue sources (brain and
HeLa). To measure the consistency of the MessageAmp II and the
Affymetrix standard protocol, we plotted the possible replicates
of the ratios for both protocols. Since there are two replicates
of input RNA from brain and two from HeLa, for each protocol
there are 4 possible ratios: 2 sets of 2 contain unique arrays.We
compared the consistency of MessageAmp II and Affymetrix ratio
estimates in Figure 6. There is generally good correspondence
(higher correlation in the log2 ratios) of one replicate versus
another in the MessageAmp II arrays relative to the Affymetrix
arrays.
|
|
|
Figure 6. Scatter
Plot of log2 Ratios of HeLa vs. Brain for MessageAmp™
II and Affymetrix Standard Protocol. Expression
values were estimated using RMA. Log2 (HeLa/Brain) are
plotted for each protocol represent the comparisons possible
where the ratios are derived from unique arrays. HeLa
and brain RNAs were amplified as stated in the MessageAmp
II and Affymetrix protocols.
|
These two analyses (Figures 5 and 6) indicate that the two protocols
are reproducible and comparisons made between the protocols do
not appear to have any systematic differences above the observed
variation between replicates within a protocol.
Present Absent Call Concordance. We used two comparisons to
evaluate Present/Absent call concordance: 1) the correspondence
of Present (P) and Absent (A) calls comparing the MessageAmp
II and Affymetrix standard protocols to address consistency between
protocols, and 2) the correspondence within the MessageAmp II
and Affymetrix protocols. The P-A calls are based on the Wilcoxon
signed rank-based gene expression Present/Absent detection algorithm
first implemented in the Affymetrix Microarray Suite, Version
5. This was done separately by RNA type (brain and HeLa). We
paired arrays in all possible combinations and summarized the
average performance. When comparing two arrays, the percent shared
Present (PSP) calls is the total genes called present in both
arrays divided by the total number of (unique) genes called present
between arrays. An equivalent statistic was used for the percent
shared Absent (PSA) calls; for this analysis we ignored Marginal
(M) calls. Also included was total concordance, calculated using
all call types by simply dividing the number of concordant calls
(P-P, A-A, or M-M) by the total number of calls (or genes = 8793).
The results, shown in Figure 7, demonstrate again that the correspondence
between the MessageAmp II and Affymetrix protocols was equivalent
to that seen within Affymetrix replicates.
|
|
|
Figure 7. Comparison
Between and Within Protocols of Mean Percent Shared Present
and Absent Calls. Percent
shared present (PSP) calls is the total genes called
present in both arrays divided by the total number of
(unique) genes called present between arrays. An equivalent
statistic was used for the percent shared absent (PSA)
calls; for this analysis we ignored marginal (M) calls.
The right most column contains the total concordance,
which is the number of concordant calls (P-P, A-A, or
M-M) divided by the total number of calls (or genes =
8793). (Present/Absent calls were determined with Affymetrix
GeneChip® operating software (GCOS) for all arrays.)
|
IVT incubation time vs. Microarray Signals
While a shortened
IVT time may produce sufficient cRNA for microarray hybridization,
the signal characteristics of the labeled cRNA on a microarray
is the critical factor for determining functionality. A series
of three IVT time points (4, 6, and 14 hr) using total RNA from
brain and HeLa were tested to measure whether any difference
was observed between IVT times. Total RNA input amounts used
were 100, 200 and 1000 ng. These samples were amplified and labeled
as above, then hybridized to GeneChip Human Genome Focus
Arrays. Again, for a reference series we used the Affymetrix
protocol with duplicate 1000 ng reactions of the same RNA. Fourteen
arrays were scanned and signal intensities were analyzed for
similarity.
We first measured the simple correlation of
the log2 expression of each array versus the chosen standard
(1000 ng input; 4 hr IVT). Figure 5b lists the correlation and
standard deviation (SD) of the differences with the standard.
This data is also expressed as simple plots of log2 (expression)
of each array versus the standard (x axis) (Figure 8). These
comparisons reveal that the magnitude of the variation one sees
among the different IVT times using 1000 ng RNA inputs and the
MesageAmp II protocol is similar or less than expected in replicates
using the Affymetrix protocol. Additionally, arrays with
cRNA from 200 ng total RNA have more under-expressed genes (a
broader tail on the left of each plot) for all times measured.
This range of differences appears to be consistent with that
also seen in Affymetrix replicates (Figure 5b). While the standard
deviation (SD) of the differences between the 200 ng RNA inputs
appears twice as large, combining replicates with simple averaging
should decrease the SD.
|
|
|
Figure 8. Expression
by IVT Time and RNA Input vs Standard. Log2
expression by IVT time and RNA Inputs (1000 ng and 200
ng) from IVT reactions incubated at 4, 6, or 14 hours
were plotted versus the Standard (4 hr IVT; 1000 ng
RNA input). The X and Y axis represent the log2 of the
expression values estimated using RMA.
|
Expression Analysis at Lower RNA Inputs
Lower RNA inputs (below
100 ng), which generally require two rounds of amplification,
suffer both a decrease in the percent Present calls and an increase
in noise. Several studies have characterized and compared single
vs. double round amplification [7, 8]. While possible, interpretation
of expression profiles between samples that have been processed
differently (single vs. double round amplification) is more problematic.
Nevertheless, we attempted to measure the difference observed
as the input levels are lowered to 10 ng of total RNA (approximately
1000 cell equivalents).
This experiment consisted of two variables: RNA input (1000,
100, and 10 ng), and amplification protocol (MessageAmp II vs.
Affymetrix). For this analysis, both the 100 and 10 ng samples
underwent two rounds of amplification.
Note that while the goal of the study was to
compare array data generated after two rounds of amplification,
MessageAmp II yields from one round of amplification with 100
ng inputs was actually sufficient for array hybridization. In
this case, a single round of amplification would be preferred,
as it would obviously improve correlation.
Again, we used 1000 ng input RNA as the standard to which we
compared the other RNA input data (Figure 9A). Scatter plots
of the log2 (expression) for both 100 and 10 ng RNA versus 1000
ng (within a protocol) is shown in Figure 9B. Obviously the 10
ng has much lower fidelity (correlation) with the standard for
both MessageAmp II and Affymetrix.
|
|
|
Figure 9. (A)
Correlation and Standard Deviation of Differences for
RNA Input. Correlation
and Standard Deviation are presented for differences
in the brain samples, when comparing inputs of 10, 100,
and 1000 ng to the standard (1000 ng). (B)
Scatter Plots of Log2 (expression) by RNA amount. Each
input is plotted vs the standard of 4 hour IVT using
1.0 µg for both MessageAmp™ II (top row) and GeneChip®
IVT Labeling Kit (bottom row). The 0.1 µg and 0.01 µg
samples are derived from two rounds of amplification.
|
As done previously, for comparison we used
the standard deviation of the differences between two replicates
of the standard (0.15 and 0.17). The results suggests that fidelity
decreases with decreasing RNA input, but particularly for the
100 ng concentration, the correlation (and standard deviation
of differences) is better for the MessageAmp II protocol than
the Affymetrix standard protocol (0.3 vs. 0.55). At 10 ng inputs
fidelity is lower. Nevertheless, the signal correlation indicates
meaningful data can still be obtained (0.91 vs. 0.93). Again,
in some cases two rounds of amplification are not needed (e.g.
for 100 ng inputs with the MessageAmp II procedure), and in this
case a comparison between the 1000 ng standard and 100 ng single
round amplification is significantly more accurate and applicable.
Conclusion
The critical criteria for microarray sample
preparation methods are: they must be highly reproducible, conserve
the original mRNA profile, and be applicable with a reasonable
range of total RNA inputs. While it is important that both amplification
and labeling methods conserve the original expression profile,
we realize that all methods will have some inherent bias [8].
Some of this bias can be minimized with a robust protocol and
optimized reagents. The MessageAmp II aRNA Amplification Kit
provides such a protocol and the novel reverse transcriptase
(ArrayScript) for this purpose. We attempted to monitor correlation and
standard deviation between two important factors, amplification
protocol and RNA input. The data indicate that the MessageAmp
II protocol is highly reproducible and can be meaningfully compared
to the Affymetrix standard protocol and the original MessageAmp
protocol (see sidebar, MessageAmp II Concordance
with Original MessageAmp Kit). We hope to have provided some guidelines for
amplification expectations and confidence that a shortened IVT
incubation time that produces enough cRNA for a microarray will
not significantly effect expression data (compared to longer
IVT times). The above examples have been an important part of
our research, production, and quality control development for
procedures that we use to maintain the highest quality reagents
for microarray analysis. We plan on continuing such studies for
both new product development and improving our line of RNA amplification
products.
Affymetrix and GeneChip are registered trademarks owned or used
by Affymetrix, Inc.
back
to top
|
