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From the Department of Pediatrics,
*
Division of Oncology/Hematology, Sophia Children’s Hospital, Erasmus University Rotterdam, Rotterdam, The Netherlands; the Department
and Central Laboratory
of Hematology, University Hospital Nijmegen, Nijmegen, The Netherlands; and Applied Biosystems, Inc.,
Foster City, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Ct2
value, is dependent on the amplification efficiency,
and is used to compensate for efficiency differences between patient
samples and the calibrator. The ß-actin reference reaction is also
used to normalize for DNA input. Furthermore, the efficiency
compensation control facilitates identification of patient samples that
are so contaminated with PCR inhibitory compounds that different
amplification reactions are affected to a different extent. Accurate
quantitation of residual disease in these samples is therefore
impossible with the current quantitative real-time PCR protocols.
Identification and exclusion of these inadequate samples will be of
utmost importance in quantitative retrospective studies, but
even more so, in future molecular diagnostic
analyses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various quantitative real-time PCR and RT-PCR protocols have recently been developed for the detection of disease associated chromosomal translocations,13, 14, 15, 16 or disease-associated T cell receptor or Ig gene rearrangements.17, 18 All protocols developed thus far are based on the assumption that patient samples and calibration samples have equal efficiencies during amplification. At least for the amplification of genomic DNA templates this assumption may not be true, since many patient samples seem contaminated with PCR inhibiting compounds. It is exactly for this reason that competitive PCRs have been developed in the past.5, 6, 7, 8 Factors like DNA fragmentation,19 residual RNA in DNA preparations,20 the presence of PCR inhibiting compounds like residual anti-coagulant heparin21 or proteinase K-digested heme compounds like hemoglobin22 or myoglobin23 will negatively affect PCR efficiency.
In this report, we demonstrate that the amplification efficiency for
different DNA templates isolated from whole blood or bone marrow
samples is subject to extensive variation, leading to underestimation
of minimal residual disease or false negative result in quantitative
PCR analyses. We developed a general real-time PCR control, ie, the
efficiency compensation control (ECC), to compensate for different
efficiencies between patient samples. This approach comprises the
inclusion of two different reference reactions, one of which is carried
out with mutant primers. Both reference reactions have been developed
to amplify with equivalent efficiencies on the same template. The exact
difference in the threshold cycle values (Ct) between both reference
reactions (Ct value) is dependent on the amplification efficiencies.
This Ct value can, therefore, be used to calculate the exact
efficiencies of both reactions for each sample in comparison to a
calibrator sample, thus providing a way to compensate for differences
in efficiencies between patient samples and the calibrator in
quantitative analyses. In this way, accurate estimation of residual
disease in patient samples becomes possible, regardless of DNA quality
or the presence of PCR-inhibiting compounds.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 | (1) |
Normalization
To correct for differences in DNA or RNA load between patient
samples, the target PCR, t(14;18) in this report, is normalized to a
reference PCR, ß-actin in this report, that is specific for an
endogenous housekeeping gene. The t(14;18) and the ß-actin reactions
outlined in this reporthave equivalent amplification
efficiencies, allowing direct normalization of the t(14;18) PCR to the
ß-actin PCR at the Ct level (Eq. 2
; K Livak, personal communication
and Applied Biosystems User Bulletin #2).
 | (2) |
PCR Efficiency
The efficiency (E) of a reaction can be
determined by a standard curve. Ct values for serial template dilutions
in dH2O are related to the logarithm of the
dilution factor, and the slope is a measure for reaction efficiency
(Eq. 3
; K Livak, personal communication and Applied Biosystems User
Bulletin #2):
 | (3) |
Quantification Using a Single Calibrator \
For quantification, patient samples can be related to a single
calibration sample, facilitating the use of Eq. 4
(ie, the comparative
Ct method; K Livak, personal communication and Applied Biosystems User
Bulletin #2). However, this method demands equivalent amplification
efficiencies between patient samples and the calibrator.
 | (4) |
Ct1 = Ct1S - Ct1C The calibrator (X0N,Cal) used for the quantitation of circulating lymphoma cells during the course of disease in follicular non-Hodgkin’s lymphoma (NHL) patients is a 100-fold dilution of t(14;18) positive cell line SUDHL6 DNA into a DNA pool obtained from four EBV-induced lymphoblastoid B cell lines, and equals 750 t(14;18)-carrying lymphoma cells per 0.5 µg of DNA (equivalent to 75,000 cells).4, 14
The Efficiency Compensation Control (ECC)
The ECC outlined in this report is used to compensate for varying
PCR efficiencies between patient samples and the calibrator. The ECC
depends on two different reference reactions, ie, the multicopy
ß-actin gene and the single copy albumin gene. Both reactions amplify
with equivalent efficiencies on identical DNA templates (see results),
but provide different Ct values. The difference between both Ct values,
ie, the Ct2 value, is solely dependent
on the actual PCR efficiency for that template (Eq. 5)
:
 | (5) |
Ct2 = Ct(ßact)-Ct(Alb)
Ct2 values can be used to determine
amplification efficiencies for patients samples relative to the
efficiency of the calibrator, providing a correction factor for
different efficiencies between patient samples and the calibrator (Eq 6)
:
 | (6) |
 | (7) |
DNA Isolation
Patient whole blood sample DNA was isolated by a standard
high-salt lysis method as described by Miller et al.25
t(14;18) PCR Analysis
Amplification reactions are performed in the ABI/Prism 7700
sequence detector (Applied Biosystems, Foster City, CA). Five hundred
nanograms of patient sample DNA is amplified in duplicate in the
presence of 300 nmol/L oligonucleotides MBR-2 (5'-TCC CTT TGA CCT TGT
TTC TTG A-3'; bp 2816–3037 of GenEMBL database entry acc. 14745) and
JH-con,1
160 nmol/L of dual-labeled fluorogenic MBR
internal probe 5'-(FAM)-CAC AGA CCC ACC CAG AGC CC-(TAMRA)-p3', 250
µmol/L dNTPs, 1.25 U of AmpliTaq DNA polymerase and 4
mmol/L MgCl2 in Sample Buffer A (Applied
Biosystems) in a volume of 50 µl. Samples are heated for 10
minutes at 95°C and amplified for 50 cycles of 15 seconds at
95°C and 60 seconds at 60°C.
The Efficiency Compensation Control
Five hundred nanograms of patient sample DNA is amplified in
duplicate in the presence of 100 nmol/L ß-actin forward and reverse
primers,7
100 nmol/L of dual-labeled fluorigenic ß-actin
probe,7
300 nmol/L mutant albumin forward (5'-TGA AAC ATA
CGT TCC CAA AGA GCC T-3') and reverse (5'-GAG AGG AAG AGT CTT TCA CAC
GTA GA-3') primers, 100 nmol/L of dual-labeled fluorigenic albumin
probe (5'-(JOE)-TGC TGA AAC ATT CAC CTT CCA TGC AGA-(TAMRA)-3'), 250
µmol/L dNTPs, 1.25 U of AmpliTaq DNA polymerase, and 4
mmol/L MgCl2 in Sample Buffer A in a total volume
of 50 µl. Amplification conditions are the same as mentioned above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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.We then determined the efficiencies for both reactions on patient DNA
samples that were isolated from lymph node biopsies or whole blood
specimen (Table 2)
. Lymph node DNA samples most likely seem to contain
higher contents of PCR inhibiting compounds resulting in lower
amplification efficiencies compared to patient whole blood DNA samples.
Both reaction efficiencies for each DNA sample compared, showed that a
low efficiency for one reaction was accompanied by a low efficiency for
the other reaction. For lymph node DNA samples the average efficiencies
for the t(14;18) and albumin reactions are E = 0.66 and
E = 0.72 respectively, but are E = 0.94
and E = 0.94, respectively, for whole blood DNA
samples. The average efficiency difference (E value)
between both reactions is E = 0.023 for lymph node
DNA samples (n = 3 pairs) and
E = -0.009 for whole blood DNA samples
(n = 8 pairs).
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, we have developed the "efficiency
compensation control (ECC)". This control uses two reference
reactions that are specific for the ß-actin and the albumin
housekeeping genes. When testing both reaction efficiencies on serial B
cell line DNA pool dilutions (DNA pool from four EBV-induced B cell
lines), both reactions performed with efficiencies of E = 0.95 and E = 0.94 for the ß-actin and albumin
reaction, respectively. ß-actin is a multicopy gene with several
pseudogenes and albumin is a single copy gene, and are present in a
constant ratio for genomic DNA templates. Both PCRs will therefore
produce different Ct values at identical template concentrations
(Figure 1)
. For genomic DNA templates, the Ct value between these reactions
(Ct2 value) is exclusively determined by the
actual amplification efficiencies for both reactions (Figure 1)
, and is
independent of initial DNA concentration. In the experiment as
described above, the Ct2 value between the
ß-actin reaction and the albumin reaction remained constant over all
serial B cell line DNA pool dilutions (500 ng - 8 ng) and was
Ct2 = 3.86 ± 0.08. Samples that amplify
with high reaction efficiencies will provide lower
Ct2 values in comparison to samples that are
contaminated with PCR inhibiting compounds and consequently amplify
with lower reaction efficiencies (Figure 1)
. This
Ct2 value can therefore be used to compensate
for efficiency differences between patient samples and the calibrator
in quantitative minimal residual disease studies. The target and
reference PCRs should be performed on the same template dilution to
exclude differences in reaction efficiencies.
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Ct2 value that is
theoretically needed between two reference PCRs to distinguish between
efficiency differences as low as E = 0.05. This will of
course depend on the reproducibility of both Ct values for the
reference reactions. The standard deviations in Ct values for both
reference reactions performed on the serial B cell line DNA pool
dilutions as described above ranged between 0.007 cycles for 0.5 µg
DNA to 0.11 cycles for the highest dilution (0.8 ng DNA). Assuming a SD
in Ct values of 0.11 cycles for both reference reactions, the
Ct2 value between both reactions needs to be
in the order of ten cycles to distinguish between efficiency
differences among DNA samples as low as E = 0.05. No
combinations of multicopy gene PCRs (ß-actin, 18S ribosomal DNA)
versus single copy gene PCRs (albumin, RNase-P) were
identified that produced a sufficient Ct2
value. As an alternative, a mutant albumin primer pair was designed
that amplified with an efficiency of E = 0.96, compared
to the ß-actin PCR (E = 0.95 as described
above). The mutant albumin primer pair produced Ct values that were
about 6 cycles higher Ct values for each template dilution in
comparison to the wild-type albumin reaction. The
Ct2 values between the ß-actin PCR and this
mutant albumin PCR was about 9 cycles over all template dilutions
tested. This PCR combination may work for the Efficiency Compensation
Control. To minimize variation in Ct values for both reactions, a
multiplex PCR was developed. Using 100 nmol/L of ß-actin primers and
300 nmol/L of mutant albumin primers, both reactions produced Ct values
of 19.98 ± 0.10 and 30.06 ± 0.03, respectively, in a
multiplex setting, and Ct = 19.25 ± 0.01 and 27.62 ±
0.04, respectively, in separate PCRs.
The ECC was tested in a retrospective study on 47 selected blood and
bone marrow DNA samples obtained from 12 follicular NHL patients that
had been isolated between 1990–1994 using the high-salt-lysis
method.25
Two of these samples had previously demonstrated
severe PCR inhibition as assessed by conventional PCR (not shown). PCR
inhibition was not due to DNA degradation as determined by gel
electrophoresis (not shown). When performing the ECC multiplex PCR,
thirty-two samples produced reasonable Ct values for both reference
PCRs with Ct2 values (range
Ct2 = 8.19–9.88; mean
Ct2 = 9.04). Assuming an average amplification
efficiency of E = 0.94 for these samples (Table 2)
, the
efficiency for each sample was then calculated based on the
Ct2 value and ranged between E = 0.83 and E = 1.07 (equation 6)
. Fifteen remaining
patient samples (32%) produced Ct2 values
lower than Ct2 = 8.19
(Ct2 = -6.83 to Ct2
= 7.82), and theoretically correspond to abnormal efficiencies of
E = 1.15 and higher. These samples may suffer from
severe PCR inhibition that affects both reference PCRs to a different
extent, and can therefore not be corrected for different efficiencies
using the ECC. Analysis of Ct2 values from
freshly isolated patient DNA samples in a prospective study revealed
that 2 out of 50 samples (4%) suffered from this same problem (not
shown).
The Ct2-based efficiencies were compared to
efficiencies that were calculated from standard curves. Four samples
with high Ct2 values (low efficiency) and four
with low Ct2 values (high efficiency) were
selected to construct standard curves. For seven out of eight samples,
the ECC determined efficiencies match the efficiencies as determined
from the standard curves of the t(14;18) and albumin PCR (Table 3)
.For five samples, the ECC based efficiencies fall within the SD of the
efficiencies that were derived from standard curves.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Testing the efficiencies for the t(14;18) target and albumin reference
PCR for various patient DNA samples, efficiencies for both reactions
varied between different patient samples probably due to contaminating
PCR inhibiting compounds. PCR efficiency was not affected by amplicon
size (Table 2)
, at least for amplicons ranging between 89 and 520 bp,
nor was it due to DNA degradation. DNA templates isolated from patient
lymph node biopsies amplify with lower efficiencies in comparison to
patient whole blood DNA samples (Table 2)
. Most importantly, for each
DNA sample the efficiencies for both PCRs were equivalent, indicating
that a low t(14;18) reaction efficiency for a DNA template is
accompanied by a similar low efficiency for the albumin reaction for
that same template. This indicates that PCR inhibiting factors affect
different reactions performed on the same template to a similar extent.
For all t(14;18) and albumin standard curves, the deviations of Ct
values for all template dilutions relative to the regression curve were
equivalent, and no evidence was found for increasing deviations at
higher template dilutions as should be expected in case of dilutable
PCR inhibition (all r2 values exceeded
0.98 but in general were in the order of 0.995). PCR inhibition
for the samples evaluated in this study was non-dilutable, and is
presumably caused by compounds that tightly bind to the DNA like
hemoglobin degradation compounds.
Our results show that quantitative studies tend to suffer from
efficiency differences between samples, leading to inaccurate results.
For instance, lower reaction efficiencies for patient samples resulting
in higher Ct values will lead to underestimation of residual disease
content. This especially affects patient samples with low residual
malignant cell numbers. Normalization of the target PCR (the t(14;18)
reaction in this report) to a reference PCR, usually specific for an
endogenous housekeeping gene, will only partially compensate for this
problem since the number of reference gene copies is always relatively
high in comparison to the number of target copies reflecting the amount
of malignant cells. The efficiency compensation control (ECC) was
developed to compensate for sample specific efficiency variation in
comparison to a calibrator (equation 7)
, and consists of a multiplex
PCR for two endogenous house keeping genes, ie, ß-actin and albumin,
that are present in a constant ratio in DNA templates but that produce
different Ct values (Ct2) for identical
template. Both reactions performed with equivalent efficiencies on
serial template dilutions, and it is reasonable to assume that both ECC
reference reactions will be equally affected by contaminating PCR
inhibiting substances as is the case for the t(14;18) and wild-type
albumin PCR. The Ct2 value is therefore
independent of initial template concentration, but depends on the exact
amplification efficiencies (equation 6) . The ECC can theoretically
distinguish between patient samples that differ in amplification
efficiencies as low as E = 0.05. Disease associated
chromosomal aneuploidy involving the ß-actin or albumin genes may
affect results obtained by the ECC. This is unlikely for follicular NHL
blood or bone marrow samples since the total number of circulating
t(14;18) positive lymphoma cells during the course of disease is in
general below five percent of total mononuclear
cells.3, 4, 8
However, for leukemias with a high tumor cell
percentage in blood and bone marrow samples during apparent disease in
contrast to clinical remission phases, this could be a problem.
Patient DNA templates that had been stored for several years produced
abnormal ECC Ct2 values for 32% of the
samples compared to 4% of freshly isolated patient DNA samples. For
these samples, the ECC will not be useful to correct for efficiency
differences in comparison to a calibrator sample. These samples
probably contain so many PCR inhibiting compounds that different PCRs
are affected to a different extent. Quantitation of residual disease in
these samples will therefore not be possible. The ECC seems very useful
in identifying such patient samples in quantitative analyses.
In conclusion, the ECC serves three purposes: first, the ß-actin Ct
value for each sample is used to normalize for different DNA input.
Second, the ECC Ct2 value is used to
compensate for efficiency difference in comparison to the calibrator.
Third, the ECC identifies patient DNA templates that may not be
quantifiable for minimal residual disease content due to unequal
inhibition of the various amplification reactions. Since the ECC uses
two reference PCRs that are specific for endogenous house keeping
genes, it may be applicable as a general control in real-time
quantitative studies using genomic DNA templates. In our opinion, the
ECC approach is a powerful control for TAQMAN-based analyses that can
be performed in the ABI PRISM 7700 system (Applied Biosystems) but
needs further evaluation in other real-time PCR systems. For probe
designs, other fluorescent reporter groups can be used, but FAM in
combination with JOE or VIC proved to be a useful combination in a
multiplex setting. Hydrolyzing probes may be replaced by molecular
beacons or scorpion primers.26
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Acknowledgments |
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Footnotes |
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Accepted for publication February 23, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 F. Dijk, E. Kraal-Muller, and W. Kamphuis Ischemia-Induced Changes of AMPA-Type Glutamate Receptor Subunit Expression Pattern in the Rat Retina: A Real-Time Quantitative PCR Study Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 330 - 341. [Abstract] [Full Text] [PDF]
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A. Tichopad, M. Dilger, G. Schwarz, and M. W. Pfaffl Standardized determination of real-time PCR efficiency from a single reaction set-up Nucleic Acids Res., October 15, 2003; 31(20): e122 - e122. [Abstract] [Full Text] [PDF]
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T. Bar, A. Stahlberg, A. Muszta, and M. Kubista Kinetic Outlier Detection (KOD) in real-time PCR Nucleic Acids R |
