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Molecular Diagnostics and Genetics |
1
Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
2
Laboratory of Analytical Chemistry, Department of
Chemistry, University of Athens, Athens 15771, Greece.
a Author for correspondence. Fax 519-973-7098; e-mail tkc{at}uwindsor.ca.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Reverse transcriptase polymerase chain reaction (RT-PCR)1 provides highly sensitive detection of mRNA sequences and has been used widely for the study of gene expression in malignant and physiological states (5)(6). In 1992 Moreno et al. (7) applied RT-PCR methodology to the detection of prostate-specific antigen (PSA) mRNA as a specific marker of circulating prostatic cells in patients with metastatic prostate cancer. In 1994 Katz et al. (8) first applied RT-PCR for PSA mRNA in the preoperative staging of prostate cancer patients (molecular staging). In recent years, the analytical sensitivity of the technique has been improved substantially by increasing the number of PCR cycles, using nested PCR, using hot-start PCR, or enhancing the detectability of the amplification products (4)(9)(10)(11)(12)(13). Several clinical studies have also been performedusing peripheral blood (4)(9)(10)(11)(12)(13), lymph nodes (14), or bone marrow (15)(16) from prostate cancer patients.
Most of the PSA mRNA assays reported previously have been qualitative; that is, they detect the presence or absence of the particular mRNA without referring to its quantity. This has produced considerable controversy regarding the usefulness of the RT-PCR test in staging prostate cancer. For example, highly sensitive RT-PCR protocols have led to the detection of PSA mRNA in the blood of healthy individuals and patients with hyperplasia (17)(18)(19)(20). The ability of circulating tumor cells to cause metastasis is still controversial, and a relationship between PSA mRNA concentration and metastatic potential has not been reported. Several reports have addressed the need for a quantitative assay for PSA mRNA (13)(17)(18)(19).
The limited number of reported quantitative protocols for PSA mRNA have been based on the parallel amplification of PSA mRNA with the mRNA of ß-actin, a housekeeping gene (21)(22)(23). A crucial assumption of the entire procedure is that the housekeeping gene has an even rate of transcription and is independent of different degrees of cellular activation. However, it has been reported that ß-actin mRNA concentrations increase with the malignant transformation of cells (24). In addition, the efficiency of reverse transcription and amplification for PSA and ß-actin mRNA may vary, thus limiting the usefulness of the technique in a clinical setting.
In this work, we developed a quantitative analytical methodology for PSA mRNA that is based on the coamplification of the target RNA with a recombinant RNA internal standard (IS). The IS has the same primer binding sequences as the PSA mRNA and both amplification products are the same size, differing only in a 24-bp centrally located sequence. The RT-PCR products are captured on microtiter wells and analyzed by two separate nonradioactive and highly sensitive hybridization assays, using specific probes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The following oligonucleotide sequences were used in the course of this
work: (i) 5'-(NH2)-CTC TCG TGG CAG GGC AGT
CT-3', a 20-mer used as the upstream primer (u) in quantitative PCR
homologous to a sequence in exon 2 of the PSA gene;
(ii) 5'-GGT CGT GGC TGG AGT CAT CA-3', a 20-mer used as the
downstream primer (d) in quantitative PCR complementary to a sequence
in exon 3 of the PSA gene (22); (iii)
5'-CTT GCT GAA CTT CTG ACT ACG ACT TGG GCA GCT GTG AGG-3',
a 39-mer (a) used as the downstream primer for synthesis of short
product A; (iv) 5'-AGT CGT AGT CAG AAG TTC AGC
AAG CTT GCT GGG TCG GCA-3', a 39-mer (b) used as the upstream
primer for synthesis of short product B; (v) 5'-CTA
ATA CGA CTC ACT ATA GGG CTC TCG TGG CAG GGC A-3', the 37-mer
upstream primer (T7-u) homologous to PSA exon 2 and bearing
the T7-promoter sequence (in italics); (vi)
5'-ATC ACG CTT TTG TTC CTG ATG CAG-3', the 24-mer probe
(p1) used in the hybridization assays of amplified target
RNA sequences. The probe spans the exon 2/exon 3 junction in the PSA
mRNA with a complementary sequence to the last 12 bases of exon 2 and
the first 12 bases of exon 3 of the PSA gene.
(vii) 5'-CTT GCT GAA CTT CTG ACT ACG ACT-3', the 24-mer
probe (p2) used in the hybridization assay of the amplified
RNA IS. The underlined segments in primers (a) and (b) represent the
new sequence to be introduced in the internal standard and are
complementary to one another. The relative positions of primers and
probes are shown in Fig. 1
. The oligonucleotides were synthesized by Bio-Synthesis.
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rna isolation
We sedimented 10 LNCaP cells by centrifugation
for 2 min at 12000g and added 1 mL of Trizol LS reagent
(25). Total RNA was then isolated according to the
manufacturer's instructions. RNA, precipitated with 2-propanol, was
washed with 750 mL/L ethanol and redissolved in 20 µL of
diethylpyrocarbonate-treated water containing 37 units of RNase
inhibitor.
Whole blood (5 mL), used for isolation of total RNA from healthy cells, was separated with Polymorphprep according to the manufacturer's instructions. Polymorphonuclear cells were washed once in phosphate-buffered saline (10 mmol/L sodium phosphate, 1.76 mmol/L potassium phosphate, 0.14 mol/L NaCl, and 2.7 mmol/L KCl solution, pH 7.4), pelleted and resuspended in 1 mL of phosphate-buffered saline. The cells were pelleted at 12000g for 2 min. Trizol LS reagent was then added, and total RNA isolation was carried out as described above for the LNCaP cells. The RNA concentration was determined by its absorbance at 260 nm with the Shimadzu UV-160 spectrophotometer.
synthesis of the rna is
A DNA template was first synthesized, starting with the
recombinant plasmid pA75 (26), which contains a 1.4-kb PSA
cDNA insert. Two separate PCRs were set up to create two short
products, A and B, each containing a newly introduced sequence of 24
bp. For PCR-A, we used oligonucleotides (u) and (a) as upstream and
downstream primers, respectively (Fig. 1
). The downstream primer (a)
contains a 15-bp sequence at the 3' end, necessary for binding to the
cDNA, and a 24-bp extension at the 5' end. Thus, the amplification
product A consists of a 66-bp segment identical to the starting DNA and
a 24-bp addition. For PCR-B, oligonucleotides (b) and (d) were used as
upstream and downstream primers. The upstream primer (b) contains (at
the 3' end) a 15-bp sequence complementary to the cDNA and a 24-bp
extension at the 5' end. Product B consists of a 143-bp segment
identical to the starting DNA plus the 24-bp extension. The PCR mixture
(total volume, 100 µL) consisted of 20 mmol/L ammonium sulfate, 75
mmol/L Tris-HCl (pH 8.8), 1 mL/L Tween-20, 2.5 mmol/L
MgCl2, 20 µmol/L of each dNTP, 50 pmol of each primer,
1.6 x 10 molecules of plasmid DNA, and 2.5 units of
Taq polymerase. The mixtures were layered with mineral oil and placed
in the 48-well Perkin–Elmer DNA thermal cycler. The primers were added
to the mixture after the block temperature had reached 95 °C. The
cycling parameters were as follows: 95 °C for 30 s, 55 °C
for 30 s, and 72 °C for 1 min. After the completion of 30
cycles, the samples were incubated at 72 °C for 10 min and cooled to
4 °C. The products of PCR-A and PCR-B were 90 bp and 167 bp,
respectively. Twenty-microliter aliquots of each product were
electrophoresed on a 2% agarose gel, and the DNA was stained with
ethidium bromide. The 90-bp and 167-bp bands were excised from the gel,
and a small slice from each band was used in a PCR mixture similar to
the one described above but containing no primers. The mixture was
subjected to 40 cycles of denaturation (95 °C, 1 min), annealing
(55 °C, 1 min), and extension (72 °C, 2 min). Subsequently, 5
µL of this reaction mixture was amplified for 30 cycles, using oligos
(u) and (d) as upstream and downstream primers, respectively. The PCR
conditions were as described above for the short fragments. This
amplification produced a 233-bp recombinant DNA fragment.
The new DNA fragment was then fused to the T7 promoter through PCR to create a complete transcription unit. The PCR was carried out with oligonucleotides (T7-u) and (d) as upstream and downstream primers, respectively, and 1 µL of a 100-fold dilution of the previous recombinant amplification product. The cycling protocol and PCR buffer were as described above for the short fragments, with an annealing temperature of 60 °C instead of 55 °C. The products of three amplifications were pooled and purified with the Wizard PCR preps DNA purification system according to the manufacturer's instructions. Through this process, excess primers were removed, and the desired product (DNA template with the T7 promoter) was concentrated in 50 µL of water.
The RNA IS was synthesized by in vitro transcription of the T7 promoter-bearing DNA template. The transcription reaction was carried out in a total volume of 75 µL containing 40 mmol/L Tris-HCl (pH 8.0), 6 mmol/L MgCl2, 2 mmol/L spermidine, 10 mmol/L NaCl, 10 mmol/L dithiothreitol, 55 units of RNase inhibitor, 2.5 mmol/L NTPs, 200 units of T7 RNA polymerase, and 10 µL of the DNA template. The reaction was allowed to proceed at 37 °C for 90 min. The RNA was purified with Trizol LS reagent, dissolved in 20 µL of diethylpyrocarbonate-treated water, and then diluted with 180 µL of 1 g/L yeast tRNA containing 37 units of RNase inhibitor. The resulting stock RNA IS solution was treated with RQ1 RNase-free DNase. To 20 µL of stock RNA IS was added 1 µL (1 unit) of DNase and 2 µL of a 10 mmol/L MgCl2, 500 mmol/L Tris-HCl (pH 7.5) solution. After a 15-min incubation at 37 °C, the DNase was inactivated by heating at 75 °C for 5 min. Various dilutions of the RNA IS were prepared using a solution containing 0.1 g/L yeast tRNA and 200 000 units/L RNase inhibitor as a diluent. A 3 x 10-fold dilution of the stock RNA IS was used as the RNA IS working solution.
labeling of primer and probes
The upstream primer (u) was synthesized with an amino group at the
5' end and was labeled with NHS-LC-biotin (Pierce). To 0.1 mL of primer
(10 nmol) diluted in water, we added 20 µL of 0.5 mol/L carbonate
buffer, pH 9.1, and 56 µL (5 µmol) of NHS-LC-biotin dissolved in
dimethyl sulfoxide. After a 2-h incubation at room temperature, the
biotinylated primer was purified three times by size exclusion
chromatography using Nap-5 columns. The purified primer solution was
concentrated four times by lyophilization.
The probes p1 and p2 were tailed enzymatically with multiple Dig moieties. The tailing reactions were performed in a total volume of 20 µL that consisted of 0.2 mol/L potassium cacodylate, 25 mmol/L Tris-HCl (pH 6.6), 0.25 g/L bovine serum albumin, 5 mmol/L CoCl2, 50 µmol/L Dig-11–2'-dUTP, 0.5 mmol/L dATP, 25 U of terminal deoxynucleotidyl transferase, and 100 pmol of probe. The reactions were carried out at 37 °C for 45 min and were then terminated by adding 2 µL of 0.2 mol/L EDTA. The labeled probes were used without purification.
quantification of psa mRNA
For reverse transcription, a solution (total volume, 12 µL)
containing 10 pmol of downstream primer (d), 1 µg of total RNA
(containing PSA mRNA), and 2 µL of RNA IS working solution (prepared
as described previously in this section) was heated for 5 min at
70 °C and placed on ice. Then an 8-µL aliquot of a buffer
containing 125 mmol/L Tris (pH 8.3), 187.5 mmol/L KCl, 25 mmol/L
dithiothreitol, 7.5 mmol/L MgCl2, 1.25 mmol/L of
each dNTP, and 200 U of reverse transcriptase was added to the sample.
The reaction was allowed to proceed for 1 h at 37 °C, after
which the reverse transcriptase was inactivated by heating at 95 °C
for 5 min and then chilling on ice. PCR was performed on a total volume
of 100 µL containing (final concentrations) 20 mmol/L ammonium
sulfate, 75 mmol/L Tris-HCl (pH 8.8), 1 mL/L Tween-20, 2 mmol/L
MgCl2, 50 µmol/L dNTPs, 50 pmol of each of the
biotinylated upstream primer (u) and the downstream primer (d), 2.5 U
of polymerase (Ultratherm DNA polymerase, Bio/Can Scientific), and 8
µL of the reverse transcription mixture. The hot-start protocol was
followed, in which the mixture was heated to 95 °C for 5 min, and
then the primers were added. PCR was carried out for 33 cycles of
denaturation at 95 °C for 30 s, annealing (65 °C, 30
s), and extension (72 °C, 1 min). Finally, the mixtures were
incubated at 72 °C for 10 min and then cooled to 4 °C until their
analysis by hybridization.
For the hybridization assay, opaque polystyrene microtiter wells were coated overnight at room temperature with 50 µL of 1.4 mg/L streptavidin diluted in phosphate-buffered saline. Before use, the wells were washed three times with wash solution (50 mmol/L Tris, pH 7.4, 0.15 mol/L NaCl, and 1 mL/L Tween-20) using the microtiter plate washer Model EAW II from SLT-Laboratory Instruments. Then 50 µL of PCR product, diluted 10-fold in blocking solution (10 g/L blocking reagent in 0.1 mol/L maleic acid and 0.15 mol/L NaCl, pH 7.5), was pipetted into each of four wells and incubated with shaking for 30 min. The wells were then washed as above, and 50 µL of a 0.2 mol/L NaOH solution was added. After a 20-min incubation, the DNA strand was removed by washing, as above. Each Dig-labeled probe, i.e., p1 and p2, was diluted in blocking solution to 7 nmol/L and heated to 42 °C. Subsequently, 50 µL of each probe was pipetted in duplicate into the wells containing the immobilized single-stranded PCR products. Hybridization was carried out for 30 min at 42 °C with shaking in the Amerlite shaker/incubator (Amersham). The wells were washed, and 50 µL of 750 unit/L anti-Dig-alkaline phosphatase conjugate (diluted in blocking solution) was added to each well and incubated for 30 min. The wells were then washed; 50 µL of substrate solution (1 mmol/L diflunisal phosphate, 1 mmol/L MgCl2, 0.1 mol/L NaCl, and 0.1 mol/L Tris, pH 9.1) was added to each well; and the enzymatic reaction was allowed to proceed for 30 min. Alkaline phosphatase catalyzes the hydrolysis of diflunisal phosphate to produce diflunisal. At the end of this incubation period, 50 µL of developing solution (0.4 mol/L NaOH, 2 mmol/L Tb3, 3 mmol/L EDTA, and 1 mol/L Tris) were added to each well. The plates were incubated for 1 min, and the fluorescence was measured with the CyberFluor 615 time-resolved fluorometer. Excitation and emission wavelengths were set at 337 and 615 nm, respectively. Diflunisal forms a highly fluorescent ternary complex with Tb3-EDTA in alkaline solution. The excitation light is absorbed by diflunisal and the energy is transferred to Tb3 (intramolecular energy transfer), which subsequently fluoresces.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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) with a new segment of equal size.
This was accomplished by using PCR as a synthetic tool, as previously
described (27). The PSA cDNA was amplified with primers (u)
and (a) (PCR-A). PCR-B used primers (b) and (d). Subsequently, products
A and B were mixed and subjected to cycles of denaturation, annealing,
and extension in the presence of DNA polymerase but without added
primers. Because the 5' extensions of the fragments are complementary
to each other, during the cycling process the two fragments act as
primers leading to the joining of A and B and the production of the
233-bp fragment. This fragment was then fused to the T7 promoter
(through PCR) and served as the template for synthesis of the RNA IS.
After transcription and purification, the RNA IS was treated with DNase
to degrade any traces of DNA template that may have been present.
Amplification of RNA IS aliquots by PCR and analysis by hybridization
gave no signal, confirming the absence of contamination from DNA
template. To verify the presence of the new 24-bp sequence in the RNA IS and to confirm that the amplification products from PSA mRNA and the RNA IS were distinguishable by hybridization, we performed RT-PCR on two samples containing PSA mRNA and the RNA IS. Each PCR product was then analyzed in triplicate by hybridization to both probes p1 and p2. The signals obtained from amplified PSA mRNA assayed with probes p1 and p2 were 114 990 ± 4646 and 1232 ± 86, respectively. The amplified RNA IS tested with probes p1 and p2 gave signals of 2567 ± 275 and 139 830 ± 4494, respectively. Therefore, the amplified PSA mRNA binds exclusively to probe p1, whereas the RNA IS binds only to probe p2. The fluorescence signals obtained after testing each PCR product with the noncomplementary probe reflect the nonspecific binding of the probe and the alkaline phosphatase-labeled anti-Dig antibody to the solid phase (typical readings for the assay blank). This experiment also confirmed that cross-hybridization between targets and probes did not occur and that the RNA IS solution was free of contamination from PSA mRNA and/or its amplification product (and vice versa).
The sensitivity and linear range of the two hybridization assays were
established as follows. We first prepared a stock solution of
biotinylated amplification product by pooling several RT-PCRs of the
PSA mRNA. The DNA concentration of the stock was determined by scanning
densitometry of the negatives prepared from pictures of ethidium
bromide-stained agarose gels. The pUC18 DNA fragments were used as
calibrators. The Bio-Rad Model GS-670 imaging densitometer (Bio-Rad
Laboratories) was used for quantification of the DNA bands. The
Molecular Analyst, Ver. 1.0, software was used, and the dependence of
ethidium bromide incorporation on the fragment size was taken into
account. A stock solution of amplification product for the RNA IS was
also prepared as above. After quantification, various dilutions of each
stock solution were analyzed by hybridization to probe
p1 or p2. In Fig. 2
, the fluorescence (corrected for the background) is plotted vs
the concentration of the amplified DNA. The background is defined as
the fluorescence obtained when a sample containing no amplification
product was assayed. Each data point represents the average of two
assays. Concentrations as low as 4 pmol/L (200 attomoles/well) of
amplification products from PSA mRNA and RNA IS were detected with
signal-to-background ratios (S/B) of 1.5 and 3.0, respectively. The
linear range extends up to 1000 pmol/L and 500 pmol/L, respectively.
The hybridization assay for the IS was more sensitive than the assay
for the target. The probes were designed with the same GC content and
therefore, the hybrids are expected to have the same melting
temperature. The difference in the calibration graphs of Fig. 2
is
probably because of folding of the captured single-stranded target DNA
(i.e., after the NaOH step), which might interfere with the
hybridization.
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The reproducibility (within-run) of the hybridization assay was tested by analyzing samples containing 31, 125, and 500 pmol/L of amplified DNA. The CVs were 4.2%, 5.2%, and 9%, respectively (n = 5).
The ability of the proposed analytical system to quantitate PSA mRNA
from a few PSA-expressing cells in the presence of total RNA from
healthy cells was estimated by preparing mixtures containing total RNA
representative of 4 to 3000 LNCaP cells in the presence of 1 µg of
total RNA from healthy cells (isolated from whole blood as described in
Methods and Materials). A fixed amount of RNA IS was added
into each sample. After reverse transcription and amplification, the
products were analyzed by the two hybridization assays. The
fluorescence obtained for the PSA mRNA and the RNA IS was plotted as a
function of the number of LNCaP cells (Fig. 3
). The S/B ratios for PSA mRNA corresponding to 4 and 12 LNCaP
cells were 1.6 and 2.5, respectively. The fluorescence corresponding to
the RNA IS remained constant for small numbers of LNCaP cells but then
decreased as the amount of PSA mRNA in the sample increased. This is
because of the plateau phenomenon of PCR. When the amplification is in
the exponential phase, the efficiency is constant. Because the same
amount of RNA IS is used in every sample, the amount of amplified RNA
IS, and therefore the signal, is relatively constant and independent of
the starting amount of the PSA mRNA. However, as the PSA mRNA
increases, the signal for IS decreases because of competition with the
target RNA for amplification as well as the PCR plateau effect.
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The amount of RNA IS added to each sample may affect the sensitivity and the analytical range of the technique. If a large amount of IS is added, then small quantities of PSA mRNA will not be amplified enough to generate a signal. On the other hand, a small amount of RNA IS in the presence of relatively high PSA mRNA concentrations would be undetectable after amplification. In our study, the amount of IS was optimized empirically by testing serial dilutions of the stock, such that the amplification product gave a fluorescence signal that was in the middle of the linear range of the calibration curve for the hybridization assay.
In Fig. 4
, the ratio, F/FIS of the fluorescence values
obtained for the PSA mRNA and the RNA IS was plotted against the number
of LNCaP cells. The data suggest that the quantitative assay is linear
in the range of 4 to 3000 cells. Because only 40% of the reverse
transcription mixture was amplified and only 1/20th of the PCR mixture
was used for analysis, the fluorescence signal represents amplification
product from PSA mRNA corresponding to <0.1 LNCaP cell.
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To assess the overall reproducibility (between-run) of the quantitative
RT-PCR, CV studies were carried out at three PSA mRNA concentrations in
the presence of 1 µg of healthy RNA. RT-PCRs for the three pools,
containing different concentrations of PSA mRNA, were performed four
times on different days. The CVs of the F/FIS ratios
obtained from the three pools were 16.1%, 16.2%, and 14.2%. In
addition, from the F/FIS ratios and the graph of Fig. 4
, it
was estimated that the pools contained 31, 381, and 1626 cells, with
CVs of 19.1%, 19.1%, and 17.0%, respectively.
The proposed analytical methodology for quantification of PSA mRNA has several advantages. First, the method is based on the use of a recombinant RNA IS that contains the same primer binding sites as the target RNA. Thus (in the absence of mispriming events of the RT primer to the considerably larger native RNA), any variation in the efficiency of the reverse transcription and/or the PCR step affects both RNAs equally, and the ratio of the analytical responses reflects the initial ratio of the two RNAs in the starting mixture. This is proven by the linearity and the reproducibility of the assays. Second, the PCR products are confirmed by hybridization assays performed in microtiter wells, thus avoiding electrophoresis and densitometry, as well as facilitating automation for use in the routine laboratory. Finally, time-resolved fluorometry offers the high sensitivity required for this type of analysis while eliminating the need for radioisotopic labeling of PCR products or probes.
In our opinion, the proposed method will find applications in the following areas: (a) to distinguish between clinically relevant and nonrelevant concentrations of PSA mRNA, the latter possibly occurring because of illegitimate transcription (28); (b) to provide information about the relation of PSA mRNA concentrations and recurrent disease after surgery; (c) as a powerful research tool in the study of the biology of prostate cancer and the metastatic process; and (d) PSA may be a new favorable prognostic indicator in female breast cancer, as has been found in recent years (29)(30)(31)(32). In view of these findings, we expect that the assay may find application in the monitoring of nonprostatic diseases.
One drawback of the proposed quantitative assay for PSA mRNA is that it cannot distinguish if the increased mRNA concentration is because of a large number of PSA-expressing cells (containing small amounts of the particular mRNA) or to a small number of cells containing high amounts of PSA mRNA. This point should be taken into account in the clinical evaluation of the quantitative PCR assay.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  S. Kaye Simple PCR-based Method for Synthesis of Molecular Calibrators and Controls Clin. Chem., February 1, 2003; 49(2): 325 - 327. [Full Text] [PDF]
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