Clinical Chemistry 43: 2268-2273, 1997;
(Clinical Chemistry. 1997;43:2268-2273.)
© 1997 American Association for Clinical Chemistry, Inc.
Quantification of parathyroid hormone-related protein mRNA by competitive PCR and time-resolved lanthanide fluorometry
Haiqin Rong,
Hong Ji,
Ylva Pernow,
Ulrika Sjöstedt and
Elisabet Buchta
The Endocrine & Diabetes Unit, Department of Molecular Medicine, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden.
a Address correspondence to this author at: Karolinska Hospital L1:02, Stockholm, Sweden. Fax 46-8-303458; e-mail bucht{at}enk.ks.se
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Abstract
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Using dissociation and enhancement time-resolved lanthanide fluorometry,
we have developed a quantitative competitive (QC)-PCR for measuring
parathyroid hormone-related protein (PTHrP) mRNA after reverse
transcription. A cloned PTHrP cDNA target was also modified by deletion
of 10 bp and insertion of 21 bp in the midregion of the fragment and
cloned for use as a competitor (i.e., internal standard). Two primers
spanning 362 bp of target and 373 bp of competitor were designed and
one of the primers was biotinylated. Two oligonucleotide probes, one
recognizing the target and the other hybridizing to the competitor,
were labeled with Eu chelate. Two equal aliquots of PCR products were
assayed with each probe separately in streptavidin-coated wells. After
35 PCR cycles, the competitor signal decreased exponentially
(y = e (3.74 -0.624x); r2 = 0.965)
and the target signal increased exponentially (y =
e (1.14 + 0.497x);
r2 = 0.984) when 1000 copies/tube of the
competitor and 0–100 000 copies/tube of the target DNA were added.
Log-transformed data for the ratio of target to competitor signals
(y) and the copies of the target DNA added (x)
were used for plotting the linear calibration curve (y
= 2.79+2.76x; r2 = 0.976). This
QC-PCR enables analysis of multiple samples simultaneously and can be
used to study PTHrP gene expression in malignancy and physiology.
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Introduction
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Because the polymerase chain reaction (PCR) provides extraordinary
sensitivity in detection of rare copies of nucleic acid sequence,
quantitative PCR methodology has been developed and used to study gene
expression by mRNA quantification after reverse transcription
(RT)1
(1)(2)(3)(4). To overcome tube-to-tube,
sample-to-sample, and even day-to-day amplification variations during
PCR (5)(6), competitive PCR has been widely
used, i.e., coamplification of a specific target DNA and known amounts
of a competitor DNA (7)(8)(9)(10)(11). The target and competitor
DNAs share the same primer recognition sites and must be amplified with
the same efficiency; their products are analyzed separately after PCR.
Typically, quantification is performed by comparing the sample PCR
signal of target DNA in a series of replicates with the coamplified PCR
signal of various known amounts of competitor, referred to as the
internal standard in this kind of assay (2). When Wang et
al. first described the method (1), they used serial 1:3
dilutions of both sample and internal standard; quantification of one
sample required running multiple PCR tubes simultaneously. Moreover,
the two PCR products had to be physically separated before
quantification.
In the present study, we have developed a quantitative competitive PCR
(QC-PCR) to study expression of the parathyroid hormone-related protein
(PTHrP) gene. The DNA solution hybridization assay makes use of Eu
chelate-labeled oligonucleotide probes, which are measured by
time-resolved fluorometry. The whole development process, demonstrated
in Fig. 1
, enables analysis of multiple samples by means of one
calibration curve covering a wide range (10–100 000 copies).
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Materials and Methods
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pthrp cdna
PTHrP cDNA was a gift from T.J. Martin, St. Vincent's Institute
of Medical Research, University of Melbourne, Australia. The 1103-bp
cDNA was cloned in a 2961-bp plasmid, pBluescript II KS (+/-).
competitor dna
Construction of the competitor is summarized in Fig. 2
. Two pairs of primers were designed, P-1/P-2 and P-3/P-4 (Table 1
). By use of the PTHrP cDNA as a template, two fragments of 185
bp (85–269) and 171 bp (450–280) were amplified separately and
purified by agarose gel electrophoresis. Both fragments had a terminal
sequence of 17 nucleotides, at the downstream 5'-end of the first
fragment and the upstream 5'-end of the second fragment. The end 13
nucleotides of the two sequences were complementary to each other.
After purification, these two fragments plus PCR buffer, dNTP, and Taq
DNA polymerase were applied to a PCR run for 5 cycles of 2 min at
45 °C and 2 min at 72 °C to join the two fragments first, before
the amplification primers P-1 and P-4 were added. After running for 35
cycles of 1 min at 94 °C, 1 min at 60 °C and 1 min at 72 °C,
the product was separated by agarose gel electrophoresis and the 377-bp
band was excised. This fragment was cloned in a pCRTM II
plasmid of 3932 bp by use of a TA Cloning® kit
(Invitrogen). This modified PTHrP cDNA fragment was used as a
competitor, in which 10 bp of sequence (270–279), which included the
only Sau 3AI restriction site, was deleted and a 21-bp
fragment was inserted. The modification has been confirmed by
sequencing the competitor DNA with a SequenaseTM Version
2.0 DNA Sequencing kit (USBTM; US Biochemical Corp.).
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Figure 2. Construction of competitor DNA for QC-PCR.
a. Based on the sequence of cDNA of PTHrP, two pairs of
primers (P-1 and P-2; P-3 and P-4) were designed. b. After
PCR, two DNA fragments were generated, F-1 and F-2. c.
Because two terminal sequences (T-a and T-b) of F-1 and F-2 were
complementary to each other, the two fragments could combine in the
PCR. In addition, the hybridized sequences formed a 21-bp insert.
d. The joined fragment amplified with P1/P4 was cloned in
pCR II. The doubly underlined basepairs denote the
restriction site of Sau 3AI.
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View this table:
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Table 1. Primers for constructing competitor DNA and for performing
PCR, and probes labeled with Eu-chelate for detecting PCR products (5'
to 3').
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eu-chelate-labeled oligonucleotide probes
Two oligonucleotide probes were designed (Table 1
), one
recognizing the target DNA (T-probe) and the other recognizing the
competitor (C-probe). Custom oligonucleotide synthesis was performed by
Genemed Biotechnologies with an Amino Linker C3 at the 5'-end.
HPLC-purified oligonucleotides were dissolved in distilled water and
vacuum-dried in aliquots of ~50 µg. A Delfia®
Eu-Oligolabeling kit (Wallac Oy) was used for labeling. In brief, the
labeling reagent, Eu-chelate of
4-[2-(4-isothiocyanatophenyl)ethyl]-2,6-bis[N,N-bis(carboxymethyl)aminomethyl]pyridine,
was dissolved in distilled water and added to the dried oligonucleotide
at a molar ratio of 60:1 for Eu-chelate:oligonucleotide. The labeling
reaction solution was adjusted to pH 9.8 with 0.1 volume of 1 mol/L
sodium carbonate in a final volume of 50 µL (oligonucleotide ~1
µg/µL) and incubated for 20 h at room temperature. The
Eu-labeled oligonucleotide probe was purified on a column of Sephadex
G25 (Pharmacia). The pooled fractions containing the Eu-probe were
concentrated by centrifugation in a Centricon®-3
concentrator (Amicon) and aliquoted and frozen at -20 °C. After
characterization, the specific activity (per nanogram of Eu-oligo) of
the T-probe was 13.4 x 106 fluorescence counts/s;
that of the C-probe was 9.6 x 106 counts/s.
pcr and extraction of pcr products
PCR amplification was carried out with a programable thermal
controller PTC-100–96V (MJ Research, Watertown, MA) and a reaction
volume of 25 µL: 0.5 µmol/L each for upstream and downstream primer
(Table 1
), 2.5 µL of competitor DNA (103 or
104 copies), 2.5 µL of target DNA (known amounts of the
cloned PTHrP cDNA or the RT mixture), 10 mmol/L Tris-HCl, 50 mmol/L
KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP, and 0.65 U of Taq
DNA polymerase (PCR Master Reagent; Boehringer Mannheim). The cycling
profile was 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C
for 35 cycles.
The separately amplified products of target and competitor DNA were
purified by gel electrophoresis in agarose of low-melting temperature
(Boehringer Mannheim). The bands at 362 and 373 bp were excised and
extracted by a WizardTM PCR Purification kit (Promega). The
two fragments were used for testing the detection limit and specificity
of the T-probe and the C-probe, respectively.
solution hybridization assay
The hybridization assay was performed as illustrated in Fig. 3
. After PCR, two equal portions (10 µL) of product were
pipetted into streptavidin-coated microtitration strip wells (Wallac
Oy); 90 µL of Delfia assay buffer (per liter, 50 mmol of Tris-HCl,
0.15 mmol of NaCl, 0.5 g of NaN3, 5 g of
bovine serum albumin, 10 mg of diethylenetriamine pentaacetic acid, and
0.1 mL of Tween 40) supplemented with 1 mol/L sodium chloride, was
added to each well. After incubation with shaking for 1 h at room
temperature, the wells were washed once with 300 µL of Delfia washing
buffer [25-fold dilution of NaCl 225 g/L, Tris-HCl 125 mmol/L, Germall
II (Sutton Labs., Chatham, NJ) 25 g/L, and Tween-20 1.25 g/L, pH 7.2];
100 µL of denaturing solution (125 mmol/L NaOH, 125 mmol/L NaCl) was
then added to each well and incubated with shaking for 5 min. After
three washes, the PCR products were hybridized for 1 h at room
temperature with Eu-labeled probes diluted in the assay buffer, one
portion with T-probe and another portion with C-probe (~30 ng of
Eu-T-probe and 40 ng of Eu-C-probe per well). After six stringent
washes, 100 µL of Delfia Enhancement Solution (Wallac Oy) was added.
The plate was incubated with shaking for 30 min, and the fluorescence
was measured by a Delfia 1234 plate fluorometer (Wallac Oy).
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Figure 3. Process for quantification of the competitive PCR (C-PCR):
(a) Target DNA, competitor DNA (with black
bar), and two primers (one biotinylated,
B—) are applied to C-PCR;
(b) after C-PCR, two equal aliquots of the
products are pipetted into streptavidin
(SA)-coated wells; (c)
denaturing solution is added and one strand of DNA is washed away;
(d) Eu-labeled T-probe is added to one portion of
the products and Eu-labeled C-probe (with black bar) is
added to another portion; (e) after
hybridization, the excess probes are washed away and Delfia Enhancement
Solution is added; (f) the fluorescence signal
from the dissociated and rechelated europium ions is measured by
time-resolved fluorometry.
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calculation
The target and competitor fluorescence signals in each PCR tube
were used to calculate the target/competitor ratio (T/C ratio). All the
T/C ratio values of the standard and samples and the number of
molecular copies of the standard were log10-transformed.
Linear regression analysis of the calibration curve (from 10 to
100 000 copies) was performed by using log T/C and log PTHrP cDNA
copies in the standard. The PTHrP mRNA copies in the samples were
calculated by use of the regression equation and calibrated in relation
to copies per microgram of total RNA.
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Results
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When the same amounts of target and competitor DNA were
coamplified in one tube for various numbers of PCR cycles, the
fluorescence signal of the two products increased in parallel. The
fluorescence signal of serial (2-fold to 16-fold) dilutions of a PCR
product decreased proportionally and in parallel when detected with
T-probe and C-probe. After immobilization of the PCR products from the
target and competitor DNAs in separate streptavidin-coated wells, 5 U
of Sau 3AI restriction enzyme were added per well
and incubated at 37 °C for 1 h. Use of this restriction enzyme
should cleave the PCR product of target DNA but not the product of
competitor DNA because this unique restriction site was deleted during
construction of the competitor DNA (see Fig. 2
, the doubly underlined
sequence). The detected fluorescence signal for the target DNA product
decreased to 8% of the control, whereas 88% of the signal from the
competitor product remained. When the immobilized target and competitor
DNA products were cross-detected, only the background signal was
recorded.
The log ratio values of the two PCR products with 103
or 104 copies of competitor DNA and various amounts of
target DNA were quite similar (Fig. 4
), being between -1.0 and 1.0. In general, the values for
103 copies of the competitor were slightly higher than
those for 104 copies, as compared with the same input
target DNA. In addition, with 104 copies of competitor, the
log ratio values corresponding to the low amount of target DNA were not
adequately separated. Thus, we chose to use 103 copies of
the competitor in the QC-PCR. Linear regression analysis of the log
ratio of the two products and the log input copies of target DNA after
30 PCR cycles showed similar results when 103 copies of
competitor were used, because the log ratio remained relatively
constant, independent of the number of PCR cycles. We used 35 cycles in
our study. Fig. 5
shows a representative calibration curve as well as the
exponential alterations of the two PCR products for various amounts
(0–100 000 copies) of target DNA and a constant amount
(103 copies) of added competitor DNA.
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Figure 4. Effect of competitor DNA copies and PCR cycles on the log
ratio of the target/competitor DNA signals with various known amounts
of PTHrP cDNA (the target) from 10 to 100 000 copies coamplified with
constant amounts of competitor DNA: 1000 (top panel) or
10 000 copies (bottom panel).
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Figure 5. Representative calibration curve and linear regression
equation of the QC-PCR assayed by time-resolved lanthanide
fluorometry.
Top: Fluorescence signals of two PCR products with various
known amounts of PTHrP cDNA (target DNA) coamplified with 1000 copies
of competitor DNA after 35 PCR cycles (r2
denotes the correlation coefficient of exponential regression).
Bottom: The plotted calibration curve
(r2 denotes the correlation coefficient of
linear regression).
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Based on four measurements of a sample cDNA (diluted 1:80)
reverse-transcribed from total RNA of a lung cancer cell line (BEN
cells, a positive control for PTHrP) that contained 26.7 (±
2.9) x 106 copies of PTHrP mRNA per microgram of
total RNA, the interassay CV was <11%. The intraassay CVs were 15%,
17%, 13%, and 10% for input of 50, 200, 2000, and 50 000 copies,
respectively, of PTHrP cDNA (n = 6). The respective recoveries
from the input of 50, 200, 2000, and 50 000 copies of PTHrP cDNA were
58%, 62%, 80%, and 109% (n = 6).
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Discussion
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The quantitative competitive PCR described here provides a
sensitive method for quantifying PTHrP mRNA in low abundance after RT.
The present QC-PCR assay differs from others described previously by
enabling accurate analysis of multiple samples with a single set of
standards over a wide range instead of requiring multiple assays for
the titration of each sample (1)(2). The two
PCR products of each tube do not need physical separation and can be
measured simultaneously in different wells on the same plate by the
respective Eu-probes. The assay can even tolerate imperfect
amplification in the presence of partial inhibition because the ratio
value of the two products remains only slightly affected. In addition
to its low detection limit and high specificity, the assay also has a
broad dynamic range (10–100 000 copies in one PCR tube), takes a few
hours instead of days, and uses no radioactive labeling. In addition,
the Eu-labeled probe has a long bench-life (at least 1 year when kept
at -20 °C).
Theoretically, for competitive PCR, the target DNA yield (T) is given
by T = T0 (1 + E)n and the competitor
DNA yield (C) is given by C = C0 (1+E)n,
where T0 and C0 are the initial amounts, n is
the number of PCR cycles, and E is the amplification efficiency.
Because E and n are equal for both target and competitor in the same
tube, the final product ratio (T/C) depends only on the initial amount
of both species of DNA; i.e., T/C = T0/C0.
In the present QC-PCR, 1000 copies of the competitor DNA, the value at
the middle of the logarithmic dynamic range, was added to a dilution
series of target DNA for establishing a calibration curve. The ratio of
final yield of target/competitor should be equivalent to the ratio of
the initial target/competitor concentrations, given Morrison and
Gannon's demonstration that coamplification of different
concentrations of different targets results in retention of the initial
proportions (12). All the PCR reactions were stopped in
the plateau phase because the onset of this phase is simultaneous for
all amplicons (12) and therefore quantification with
competitor or internal standard DNA does not require exponential
amplification (13)(14).
In developing the present QC-PCR assay, we constructed the competitor
DNA by means of a splice overlap extension PCR method. This resulted in
the deletion of 10 bp and insertion of 21 bp at the midregion of the
PTHrP cDNA fragment. By deleting some material, we could design a probe
specific for detecting the wild-type PTHrP cDNA fragment; the insertion
allowed us to design a probe specific for the competitor DNA; and both
probes hybridized on the respective middle regions. In addition, the
only restriction site for Sau 3AI was removed by the
deletion and thus provided the possibility for checking the probe
specificity. The two PCR products differ in size by 11 bp and can be
distinguished by gel electrophoresis. In view of the inverse
exponential relationship between DNA template size and amplification
efficiency (15), it is important to keep the difference of
amplification size between target and competitor as small as possible.
Competitive PCR is not by itself a method for quantification; after
amplification, the PCR products have to be accurately analyzed. For
detection and analysis of amplified products, a large variety of
methods are available (16). We used a europium chelate to
label both oligonucleotide probes. The dissociated Eu3+ in
acidic condition is rechelated by another chelator, which gives a
strong fluorescence signal detectable by time-resolved fluorometry. The
Eu-chelate has been used for labeling oligonucleotide probes in several
studies, the probes in those studies having been introduced at the
5'-end of a 20- to 40-nucleotide-long tail of modified cytidines, which
enabled the labeling of as many as 20 Eu-chelates per probe
(17)(18). In the present study, we used probes
with one amino group at the 5'-ends for labeling. This single
Eu-chelate-labeled probe is sensitive enough to detect the PCR product
but results in low background.
A limitation of this QC-PCR is that the RT efficiency of the sample RNA
was not controlled in the presence of an internal RNA standard.
However, we extracted the total RNA from all the samples by
standardized procedures, and carried out RT of 1 µg of total RNA
samples in parallel by use of the same RT master solution. This could
keep the variations of RT in different samples as low as possible. In
addition, the two primers for PCR were designed on the basis of
sequences in two exons (exon 3 and exon 4 in the PTHrP gene) with an
intron of 5500 bp in between. This should minimize problems from
genomic DNA carryover contamination during RNA extraction.
In conclusion, this QC-PCR simplifies the analysis of multiple
samples and should facilitate the studies of PTHrP gene expression in
malignant and physiological states. In addition, the Eu-labeled
oligonucleotide probe is safe and sensitive and has a long bench-life.
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Acknowledgments
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The study was supported by grants from the Swedish Cancer Society
(3129), the Swedish Medical Research Council (5992), the Funds of
the Karolinska Institute, the Foundation of Åke Wiberg, the Foundation
of Loo and Hans Osterman, and the Foundation of the Family Janne
Elgqvist. We thank Pertti Hurskainen, Wallac Oy, Turku, Finland, for
providing the reagent and method for labeling the oligonucleotides.
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Footnotes
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1 Nonstandard abbreviations: RT, reverse transcription; PTHrP, parathyroid hormone-related protein; QC-PCR, quantitative competitive PCR. 
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Top
Abstract
Introduction
