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mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction
From the Department of Medicine,
*
Roswell Park Cancer Institute, Buffalo, New York; Applied Biosystems,
Foster City, California; the Department of Pathology,
University of New Mexico Cancer Research Facility, Albuquerque, New Mexico; and the Department of Oncology,
Montefiore Medical Center and Albert Einstein Cancer Center, Bronx, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, the fusion oncogene present as
a specific marker in >99% of cases of acute promyelocytic leukemia
(APL). The assay is linear over at least 5 orders of magnitude of input
DNA or RNA, and detects as few as 4 copies of PML-RAR
plasmid DNA. PML-RAR transcripts could be detected in mixtures
containing 2 to 5 pg of RNA from fusion-containing cells in a
background of 1 µg of RNA from PML-RAR-negative cells. Using 1.0
to 2.5 µg of input RNA, the sensitivity of the assay was
between 10-5 and 10-6. Furthermore,
determination of GAPDH copy number in each reaction allowed an accurate
assessment of sample-to-sample variation in RNA quality and reaction
efficiency, with consequent definition of a detection limit for
each sample assayed. Using an internal calibrator, assay
precision was high, with coefficients of variation between 10
and 20%. An interlaboratory study using coded samples demonstrated
excellent reproducibility and high concordance between laboratories.
This assay will be used to test the hypothesis that sensitive and
quantitative measurement of leukemic burden, during or after
therapy of APL, can stratify patients into discrete risk
groups, and thereby serve as a basis for risk-adapted therapy
in APL. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Themajority of APL patients harbor a translocation between chromosomes 15
and 17, with resultant fusion of the PML gene, at 15q12, with the
retinoic acid receptor (RAR) gene, at 17q22.5
This
gene fusion results in production of a leukemia-specific chimeric mRNA,
PML-RAR. In the present study, we present the technical details,
pre-clinical validation, and initial clinical application of a
sensitive, quantitative RT-PCR assay that can detect and measure the
PML-RAR fusion transcript. The assay utilizes the 5'-nuclease
technique, in which nuclease degradation of a dual-labeled fluorogenic
probe results in a measurable fluorescence signal that is detected
during the PCR process ("real-time" PCR, or TaqMan
assay).6, 7
Our data indicate that the sensitivity of this
quantitative method is at least equal to that of a published manual
RT-PCR assay for detection of PML-RAR, and that it is 100%
specific. Furthermore, the precision of this method is good, and it is
amenable to interlaboratory standardization; these features suggest
that it is ideally suited for high throughput analysis of large numbers
of clinical specimens.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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type L (long) isoform, while UF1 cells express the S
(short) isoform (Figure 1)
. The HL-60 promyelocytic cell line does not express PML-RAR. For
RNA dilution experiments, RNA was extracted from exponentially growing
cells, quantified by spectrophotometry, and mixed at dilutions
indicated in the text or figure legends. After informed consent, blood
or bone marrow was collected from normal donors or from patients with
leukemia into Vacutainer tubes containing either disodium heparin or
EDTA anticoagulant. Mononuclear cells were prepared from blood or bone
marrow by Ficoll-Hypaque density-gradient centrifugation.
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and GAPDH plasmid
standards were never taken into the RT-PCR set-up room.
Isolation of RNA and Reverse Transcription into cDNA
Total cellular RNA was isolated using either a modification of the
guanidinium acid-phenol extraction method11
(STAT-60
reagent; Tel-Test, Inc., Friendswood, TX), or one of two proprietary
column affinity procedures (RNeasy kits, Qiagen, Inc., Valencia, CA; or
RNAqueous kits, Ambion Inc., Austin, TX), in each case according to the
respective manufacturers’ instructions. RNA quality was assessed
visually by confirmation of intact 28S and 18S ribosomal bands
following agarose gel electrophoresis and ethidium bromide staining.
Before cDNA synthesis, RNA (1.0–2.5 µg) was heated at 75°C for 10
minutes and snap-cooled on ice. Two different RT protocols were used,
which differed only by the inclusion of either gene-specific primers or
random hexamers to prime first strand cDNA synthesis. Both protocols
included 1X Taq polymerase buffer II (Applied Biosystems,
Foster City, CA; final concentrations: 50 mmol/L KCl, 10 mmol/L
Tris-HCl, pH 8.3), 5 units/µl Moloney murine leukemia virus (Applied
Biosystems) or Superscript II RNase H–(Life
Technologies, Grand Island, NY) reverse transcriptase, 250 µmol/L
each dGTP, dCTP, dATP, dTTP, 3.0 mmol/L MgCl2,
and 1.6 units/µl RNase inhibitor (Applied Biosystems or Promega,
Madison, WI). Random hexamers were used at a final concentration of 5
µmol/L, and gene-specific primers at a final concentration of 0.125
µmol/L each. Sequences for the gene-specific primers were as follows:
RAR, 5'-TGTTCTTCTGGATGCTG-3'; GAPDH, 5'-ACTTGATTTTGGAGGGA-3'. The
final reaction volume was either 20 or 25 µl. cDNA synthesis was
performed at 48°C for 60 minutes, followed by an incubation at 95°C
for 10 minutes to inactivate the RT enzyme; in reactions including
random hexamers, an initial 10-minute incubation at 25°C was included
before the 48°C cDNA synthesis reaction. In the experiment shown in
Figure 4
, gene-specific primers were used for RT, dNTP concentrations
were 1 mmol/L each, and MgCl2 concentration was
7.5 mmol/L. These variations did not materially affect the sensitivity
or specificity of the assay.
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primers and probe used in this work are indicated
in Figure 1
. All probes contained a 3' TAMRA
(6-carboxy-tetramethyl-rhodamine) quencher dye and were synthesized at
Perkin Elmer. The P4 primer was synthesized at the Biopolymer facility
of Roswell Park Cancer Institute. The RAR probe (R1) was labeled at
the 5' end with a FAM (6-carboxyfluorescein) reporter fluorescent dye,
while the GAPDH probe contained a 5' JOE label. GAPDH primers and probe
were purchased from or provided by Applied Biosystems. Final
concentrations of probes and primers (Figure 1)
were chosen based on
preliminary optimization experiments (data not shown).
Standard Curve Generation
Plasmids containing the PML-RAR and GAPDH cDNAs were
constructed using standard recombinant DNA methods. Plasmid DNA was
prepared and its concentration determined using the PicoGreen
assay.12
The DNA was 10-fold serially diluted in water and
aliquots were stored at -70°C until use. Standard curves were
generated using from 4 to 4 x 107 copies of
plasmid DNA. It is emphasized that the copy numbers/µg RNA, as cited
in the text for test samples, were derived by reference to these
standard curves and do not precisely indicate the number of mRNA
molecules, since the efficiency of RT was not directly determined.
Real-Time PCR
For PML-RAR PCR, the 50 µl reaction contained 25 µl 2X
TaqMan Universal PCR Master Mix (Applied Biosystems), 2.5–5 µl of RT
product, and appropriate PML-RAR primers and probe at concentrations
listed in Figure 1
. For GAPDH PCR, the final reaction volume was either
25 or 50 µl, and generally 2.5 µl of RT product was used.
Amplifications were carried out at 50°C for 2 minutes, 95°C for 10
minutes, followed by 40 PCR cycles at 95°C (15 seconds) and 60°C (1
minute). All reactions were carried out in MicroAmp optical tubes or
MicroAmp optical 96-well reaction plates (Applied Biosystems) using an
ABI PRISM 7700 sequence detection system. Real-time PCR data were
collected using the ABI Prism 7700 sequence detection system (Applied
Biosystems) as previously published.7
Briefly, the
normalized fluorescence intensity of the reporter dye (
Rn) is
plotted against cycle number to derive a graphical representation of
the PCR reaction. The threshold cycle CT is
defined as the cycle number at which the Rn crosses a
software-generated threshold defined as 10 standard deviations above
baseline (during cycles 3–15). The CT is
linearly proportional to the logarithm of the input copy number. By
construction of an appropriate standard curve, the starting copy number
of an unknown sample relative to the standard can be derived.
Manual RT-PCR
Reverse transcription from random hexamers and first-round PCR
amplification of PML-RAR were performed essentially as described
previously,13
making adjustments to ensure that 1 µg RNA
equivalent of cDNA was used in the PCR reaction. After 40 PCR cycles, 2
µl of a 1:20 dilution of the first-round PML-RAR L-form product
was amplified in a standard 50 µl second-round, nested reaction for
37 PCR cycles, using the following primers: upstream, anchored in the
proximal portion of PML exon 6 (5'-ACAACGACAGCCCAGAAGAGGAAGT-3');
downstream, crossing the PML-RAR junction
(5'-GCTGCTCTGGGTCTCAATGGCTG-3'). PCR amplifications were performed in a
PTC-100 programmable thermal controller (MJ Research, Inc., Watertown,
MA). We found that utilization of the PTC-100 "Hot Bonnet"
attachment, performance under a layer of mineral oil, and thorough
denaturation of both rounds of PCR amplification, using
heat-activatable Taq polymerase (Amplitaq Gold, Perkin
Elmer, Inc., Norwalk, CT) were important elements in achieving high
sensitivity. The effectiveness of RNA transcription was evaluated by
using 0.1 µg RNA equivalent of cDNA from each RT reaction for PCR
amplification of GAPDH mRNA in the presence of a fixed amount of a
Mimic DNA competitor, as previously described.14
Gel
electrophoretic analysis of the second-round products was as previously
described.13
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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reverse primer, and a
probe that hybridizes to RAR sequence at the junction of the fusion
transcript (Figure 1)
. To optimize protocols for efficient cDNA
synthesis and real-time PCR, GAPDH mRNA levels were measured in
clinical samples or cell lines by reference to standard curves
generated with GAPDH plasmid DNA (Figure 2A)
. As previously reported,15
RNA isolated from blood or
marrow samples anti-coagulated with heparin appeared to contain an
inhibitor of RT or PCR (Figure 2B)
. This problem was overcome by
isolation (or "clean-up") of RNA using either of two column-based
purification protocols (Rneasy, Qiagen; or RNAqueous, Ambion) (Figure 2B)
. cDNA yield was also influenced, albeit modestly, by the type of
primer used to initiate cDNA synthesis (Figure 2C
and data not shown).
Using optimal RNA isolation procedures and plasmid-based standard
curves, the calculated number of GAPDH copies/µg RNA was
approximately 5 x 106 (±3 x
106) using RNA from Ficoll-purified normal blood
or marrow mononuclear cells (Figure 2D)
, and approximately 1.5 x
107 (±5 x 106) using
RNA from leukemic blood or marrow mononuclear cells. The number of
calculated GAPDH copies per µg of RNA from cultured hematopoietic
cell lines (HL-60, Jurkat, etc.) was significantly higher (Figure 2D)
.
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, and the CT was linearly proportional to the
logarithm of the input copy number over at least 5 orders of magnitude
(R2 > 0.99). The dynamic range
and detection limit was similar for GAPDH (Figure 2A
and data not
shown). The Y intercept reflects the theoretical
CT value at one copy of input DNA, while the
slope (S) of the linear regression curve correlates
with the efficiency (E) of the PCR reaction,
according to the following formula: E =
10-1/S -1. A PCR efficiency of 1 (slope =
-3.322) indicates that the copy number doubles in each cycle. Using
plasmid DNA as a template, the reaction efficiencies for the PR-S,
PR-L, and GAPDH probe and primer sets were generally 0.95 or higher,
indicating near optimum PCR amplification. A similar analysis was
performed using NB4 or UF1 RNA as starting material. The results
(Figure 3B)
indicate that PML-RAR transcripts could be detected
using as little as 10 pg of either NB4 or UF1 RNA, and confirm that the
assay is linear over at least 4 orders of magnitude, from 10 pg to 100
ng of input NB4 or UF1 RNA (R2
= 0.982 and 0.998, respectively). The respective efficiencies,
calculated from the slopes of the linear regression equations, were
98% and 94%, suggesting little to no decrease in PCR efficiency
despite the more complex nature of the cDNA starting material.
Additional experiments confirmed an even lower detection limit of
PML-RAR in RNA dilutions, in the range of 3 to 5 pg of NB4 or UF1
RNA admixed with control RNA (data not shown).
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, there was excellent concordance between the two laboratories, and the
sensitivities achieved (10-5 for UF1 [S
isoform] dilutions, and 10-4 or
10-5 for NB4 [L isoform] dilutions) were
similar. Finally, the PML-RAR normalized quantities (NQ, number of
PML-RAR copies per 1 x 106 GAPDH
copies), were also similar between the two laboratories (Table 1)
.
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samples
were tested on each day, as well as an internal calibrator (NB4 RNA
diluted 1:100 in HL-60 RNA), and a negative control (HL-60 RNA). All 12
HL-60 assays were negative, as expected. On each day, separate standard
curves were generated for PML-RAR and GAPDH. The results
(Table 2)
are reported as 1) the number of PML-RAR copies per µg of
starting RNA; 2) as a GAPDH normalized quantity, NQ; and 3) as a
normalized, calibrated quantity (NQC), in which
the NQ for the experimental sample is divided by the NQ for the
internal calibrator. The coefficients of variation (CVs) were lowest
(12–16%) when data were reported with reference to an internal
calibrator (Table 2)
.
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3presents the data from a direct comparison, using the same RNA
preparation, between real-time and manual RT-PCR procedures following
serial dilution of NB4 cells in HL-60 cells (or NB4 RNA in HL-60 RNA).
Although the amounts of RNA differed between the qualitative and
quantitative assays, it can be concluded that the real-time procedure
is at least as sensitive, and perhaps more sensitive, than the
optimized manual procedure in detecting PML-RAR, and that the
dilution range over which the real-time procedure becomes variably
positive is more narrow than for the manual assay. The results in Table 3
also suggest that the practical sensitivity limit of the real-time
assay is between 1 in 10-5 and 1 in
10-6, assuming a more standard input RNA
equivalent of 500 ng to 1 µg, commonly achieved with clinical
samples.
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to demonstrate the clinical utility of the assay and introduce several
concepts related to analysis of clinical samples. The detection limit
(DL) is defined as [(1/GAPDH copy number) x
106], and thus is the NQ value that would result
if one copy of PML-RAR were present in the PCR reaction (the
arbitrary 106 multiplier is the same multiplier
used to calculate the NQ values). Note that any sample with an NQ value
above the DL is considered positive (>1 copy). By definition, the DL
depends on the number of GAPDH molecules in each tested sample, and
thus is a quantitative measurement of the sensitivity achieved with
each specimen. In a small percentage of cases (ie, sample 5 in Figure 4A
), the PML-RAR NQ value, while measurable, is nevertheless below
the theoretical DL of 1 copy. These samples generally have very high
CT values (
39), and reference to the standard
curve generates a calculated starting PML-RAR copy number of <1,
which is a theoretical impossibility. While the appropriate way to
handle such data points is not clear, it seems likely that most such
samples are true positives, and that the calculated copy number of < 1 reflects quantitative imprecision at very low input copy numbers.
Proper interpretation of negative results (samples 6–8 in Figure 4
, A
and B) requires definition of a sensitivity threshold, which is based
on the number of GAPDH copies in each sample. This threshold is by
definition user-selected but should be consistently applied across all
samples (in Figure 4
, this threshold is set at 2.5 x
105 GAPDH copies, or an approximately
10-4 sensitivity). If the DL falls below this
threshold in a given specimen (samples 6–8 in Figure 4A
, and sample 7
in Figure 4B
), then the assay was theoretically capable of detecting 1
PML-RAR copy at that sensitivity threshold, and such a sample is
considered to be a true negative. Specimens with NQs of 0 but with DLs
above the sensitivity threshold (Figure 4B
, samples 6 and 8) are
considered to be false negatives. The sensitivity threshold can be
applied consistently across a large clinical dataset to ensure
exclusion of samples that do not meet minimum requirements for RNA
input and assay performance. Figure 4
also graphically demonstrates the
kinetics of clearance of PML-RAR in this patient who received
all-trans retinoic acid for induction and chemotherapy
consolidation. The eventual relapse at 2 years was clearly anticipated
by TaqMan analysis of both blood and bone marrow samples analyzed 6
months earlier, when the PML-RAR quantity was 4 orders of magnitude
less than the value observed at frank relapse.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA with relapse risk. From a technical standpoint,
cumbersome competitive quantitative RT-PCR protocols are being replaced
by assays that use dual-labeled fluorescent probes and continuous
detection of PCR amplicons in "real time."7, 17
These
TaqMan, or real-time, RT-PCR assays are high-throughput, quantitative,
and, assuming proper probe and primer design and adequate RNA input,
highly sensitive. The assays can be used to quantify serial changes in
RNA expression relative to a baseline sample or, by reference to RNA-
or DNA-based standard curves, to determine absolute starting copy
number of a specific RNA in all samples. The purpose of the current
report is to present the technical details and pre-clinical validation
of a newly developed TaqMan protocol for detection of PML-RAR in
bone marrow or blood specimens from APL patients. The assay described
here is sufficiently sensitive and specific to quantify low levels of
minimal residual disease in APL, and will be useful to test the
hypothesis that a threshold of leukemic burden can be defined that will
correlate with outcome. Indeed, we have recently applied this assay to
a large clinical dataset of over 800 APL samples, and our preliminary
results show a statistically significant correlation between the level
of PML-RAR at the end of consolidation and relapse
risk.18
(Gallagher RE, Yeap B, Bi W, Slack JL, Harrington
DP, Livak K, Appelbaum FR, Bloomfield CD, Tallman MS, Willman CW,
manuscript submitted for publication).
A major criticism of end-point RT-PCR assays is the lack of a
quantitative measure of sensitivity on a per-sample basis.
Sensitivities are generally inferred from serial dilutions of cell line
RNA, but extrapolation from such ideal mixtures to clinical specimens
is not necessarily valid, given issues such as PCR inhibitors, poor RNA
integrity, etc. Using TaqMan, the success (and by extension
sensitivity) of each individual RT-PCR reaction can be quantified by
measurement of GAPDH or other control transcript. A minimum sensitivity
level can be established that is then used for an entire data set, and
the detection limit concept can be applied to each sample to determine
whether one copy of PML-RAR was theoretically detectable in that
sample. The critical point is that sensitivity, as generally and
previously defined, has not accounted in a quantifiable way for
day-to-day differences in specimen quality and reaction efficiency that
can clearly be significant, particularly using clinical samples. TaqMan
RT-PCR can measure these differences and include them in the read-out,
removing one of the largest variables, and sources of inaccuracy, in
studies using manual or end-point RT-PCR. Thus, the power of the TaqMan
assay is that it not only provides quantitative results that allow
comparison among positive samples, but it also permits direct
assessment of the sensitivity achieved for each negative sample. For
the TaqMan assay to be accurate in determination of sensitivity for a
specific sample, the degradation rates of the target (PML-RAR) and
control (GAPDH or other) transcripts must be relatively similar. This
issue is of paramount importance to clinical application of the assay,
since blood or marrow samples are often shipped long distances, with
unavoidable delays in processing of cells. If the target mRNA undergoes
significant degradation on storage or shipment, while the control
transcript is stable, then the sensitivity of some samples would likely
be overestimated. Experiments to address this theoretical concern are
clearly required.
Additional issues that are critical to application of this assay to
large clinical datasets include assay precision and potential for
interlaboratory standardization. The precision of an assay can be
estimated by calculation of a coefficient of variation (CV), which
reflects the day-to-day differences in results of testing the same
sample. Clearly, the CV needs to be acceptably low to propose
application of a given assay to a large dataset. The CVs reported in
Table 3
(approximately 20%) compare favorably with clinically accepted
and commercially used tests, eg, for measurement of HIV RNA levels in
plasma.19
The use of an internal PML-RAR calibrator, in
addition to the use of GAPDH as a measure of RNA integrity and reverse
transcription efficiency, gave the lowest CVs (12 to 16%), and may be
a useful protocol addition if the assay is to be performed by multiple
independent laboratories.20
The results of a blinded,
interlaboratory comparison (Table 1)
indicated an acceptable degree of
agreement, both in CT values and in calculated
copy numbers for PML-RAR, suggesting that this assay can be
standardized for use by different laboratories, which is a prerequisite
for its application to a rare disease like APL.
The TaqMan, or 5'-nuclease assay, has previously been used to quantify
disease-specific transcripts from a variety of hematological
malignancies.21, 22, 23, 24, 25, 26, 27
Several groups21, 22, 26
have published data regarding detection of BCR-ABL (the
leukemia-specific fusion transcript found in chronic myeloid leukemia)
using real-time RT-PCR. Using a one-step protocol, Preudhomme et
al22
could detect 20 pg of positive control RNA from the
BCR-ABL-positive cell line K562, which translated into a sensitivity of
approximately 10-4. Mensink et
al,21
using a different primer and probe set and using a
two-step procedure similar to that reported in the current study, could
detect at least 10 copies of BCR-ABL plasmid DNA and 10 pg of K562 RNA,
for a calculated sensitivity of 1 in 105. The
TaqMan probe used to detect the BCR-ABL amplicon was, in both cases,
derived from ABL cDNA sequence and did not cross the BCR-ABL fusion
junction. Similar to our data using a probe entirely from RAR, this
approach did not lead to any appreciable incidence of false positive
results, suggesting that non-junction-specific probes provide
acceptable specificity in the TaqMan assay. These studies and others
suggest that real-time RT-PCR, assuming properly designed primers and
probes, will have sufficient sensitivity to provide valuable prognostic
information in human leukemias.
While these experiments were in progress, Cassinat et al28
published a slightly different protocol to detect PML-RAR using the
TaqMan method. Although those authors used a different PML-RAR
primer and probe set, and a different internal control mRNA
(porphobilinogen deaminase), the sensitivity, specificity, and
precision of their assay was similar to that reported here. Given the
rarity of APL, it may be desirable to agree on a "universal" primer
and probe set that can be used world-wide to quantify PML-RAR. Such
an approach would allow more ready comparison of quantitative RT-PCR
results among different APL molecular diagnostic centers, and perhaps
lead to more universal agreement about the level of PML-RAR that
correlates with poor clinical outcome. Our results, combined with those
of Cassinat et al,28
strongly suggest that "real-time"
RT-PCR methodology is sufficiently robust to accurately quantify small
amounts of PML-RAR mRNA in clinical specimens, and it is expected
that clinical application of this assay will provide important
information for the management of APL patients. However, while this
elegant technology can clearly provide a precise measurement of the
amount of fusion RNA present in a given clinical specimen, extensive
further study is necessary to formally define transcript levels that
correlate with relapse risk at specific time points.
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Acknowledgments |
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Footnotes |
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Address requests for reprints to James L. Slack, MD, Department of Medicine, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263. E-mail: james.slack@roswellpark.org.
Accepted for publication August 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fusion transcripts in patients in long-term remission of APL. Leukemia 1998, 12:1349-1354[Medline]
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