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Articles |
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Dipartimento di Biochimica e Biotecnologie Mediche and CEINGE scarl,
2
Cattedra di Gastroenterologia, Facoltà di Medicina e Chirurgia, Università di Napoli "Federico II", I-80131 Naples, Italy.
3
Cattedra di Gastroenterologia, Seconda Università
di Napoli, I-80131 Naples, Italy.
4
Facoltà di Scienze Matematiche, Fisiche e
Naturali, Università del Molise, I-86170 Isernia, Italy.
a Address correspondence to this author at: Dipartimento di Biochimica e Biotecnologie Mediche, via S. Pansini 5, I-80131 Naples, Italy. Fax 39-081-7463650; e-mail salvator{at}unina.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Methods: We studied 12 patients with hepatocellular carcinoma, 19 patients affected by chronic hepatitis C or cirrhosis, and 7 healthy controls. Aldolase A mRNA was reverse-transcribed to cDNA, which was then amplified by PCR. The amplified segments were "read" with a novel dot-blot procedure. A calibrator with the same sequence, synthesized in vitro using a T7 phage promoter, was processed at scalar dilutions in parallel to the target samples to generate a calibration curve and so quantify the target mRNA (detection limit, 0.03 amol; linearity spanning five orders of magnitude).
Results: Aldolase A mRNA was ~10-fold higher in liver biopsies from patients with hepatocellular carcinoma vs patients with chronic hepatitis C or cirrhosis, and healthy individuals. Furthermore, aldolase A mRNA concentrations were 1.2- to 21.3-fold higher in 12 liver biopsies compared with the paired surrounding cirrhotic tissue.
Conclusions: The quantitative analysis of liver tissue aldolase A mRNA differentiates between nonneoplastic chronic liver diseases and hepatocellular carcinoma, which suggests that it has diagnostic potential.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-fetoprotein (4)
and aldolase A (5), serum protein analysis scarcely
contributes to the identification of neoplastic transformation of
cirrhosis. Therefore, because quantitative analysis of specific mRNAs is the most efficient procedure for evaluating the extent of gene expression, we used this approach in a variety of clinical situations to identify metastatic cells expressing aberrant messenger species in blood (6), and to monitor minimal residual disease (7) and the effect of therapy in patients with lymphoproliferative disorders (8). The analysis of specific mRNA has also been used to identify metastatic cells in bone marrow (9) and to analyze oncogene expression in human cancer (10).
Various procedures have been devised to quantitatively analyze specific mRNAs based on the reverse transcription of mRNA species to cDNA, followed by PCR amplification of cDNA (RT-PCR). These methods are much more sensitive than Northern analysis, RNase protection assay, and in situ hybridization. In addition, they require a small amount of nucleic acids and can be used to quantify gene expression (11). However, the results of quantitative RT-PCR techniques are not consistently reproducible (12). In our laboratory, Pane et al. (13) devised a noncompetitive RT-PCR procedure in which a synthetic calibrator, obtained by in vitro transcription and with the same sequence as the target mRNA, is analyzed parallel to the target samples. This procedure is highly reproducible in quantifying several specific mRNAs from healthy tissues and leukemia cells.
We set up a semiautomated dot-blot system for the comparative quantification of the RT-PCR-amplified cDNA. The method is highly efficient and reproducible. We applied this improved procedure to the quantitative analysis of aldolase A mRNA in liver biopsies from patients with chronic liver diseases at different stages. The analysis revealed a strong resurgence of the isoenzyme in HCC that distinguished HCC patients from those with cirrhosis and chronic hepatitis C.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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rna extraction
Total RNA was extracted from tissue as described in detail
elsewhere (14). Briefly, each sample (~15 mg) was
supplemented with 3 mL of guanidinium-thiocyanate buffer. The DNA was
then sheared through a fine needle (22 gauge), and 300 µL of 2 mol/L
sodium acetate (pH 4), 3 mL of water-saturated phenol, and 600 µL of
chloroform-isoamyl alcohol solution (49:1, by volume) were added. The
samples were centrifuged at 8000g for 20 min at 4 °C,
after which the water phase was transferred into a new tube. The RNA
was precipitated twice with ethanol, and the pellet was finally washed
in 700 mL/L ethanol in water, precipitated by centrifugation,
dried under reduced pressure, and resuspended in sterile water.
The total amount of RNA was spectrophotometrically quantified at 260
nm.
synthesis of rna calibrator
The calibrator with the same sequence as the mRNA to be quantified
was synthesized as follows (15): the aldolase A cDNA, cloned
in our laboratory (16), was amplified by PCR using specific
primers (downstream, 5'-ATAACGGGCCA GAACATTG-3'; upstream,
5'-TGACCCCGGACGAGAAGAA-3'). The latter primer contained, at the 5' end,
the adjunctive sequence TAATACGACTCA CTATAGGGAGA to incorporate the T7
phage promoter at the 5' end of the amplified DNA (15). The
amplified product, once purified by low-melting point agarose, was used
as template for the RNA transcription (17). The in vitro
transcription of the aldolase A cDNA yielded ~80 µg of the specific
mRNA. Agarose gel electrophoresis with formaldehyde was used to verify
the integrity and purity of the RNA and to exclude DNA contamination.
To further verify the absence of DNA, six samples of RNA extracted from
liver biopsies were amplified for the cystic fibrosis gene analysis
(18); in all samples, the PCR failed to amplify DNA when we
used a variety of primers usually used for the analysis of
CFTR gene sequences. The mRNA was quantified
spectrophotometrically at 260 nm, and scalar dilutions (300–0.03 amol)
of the calibrator in tRNA solution were obtained and stored at
-80 °C to prevent degradation.
amplification procedures and revelation of the target product
Both the RNA samples and RNA calibrators were reverse-transcribed
into cDNA using a mixture containing, in 20 µL: 750 ng of total RNA
or the RNA calibrator (diluted), 5 mmol/L MgCl2,
50 mmol/L KCl, 1 mmol/L each deoxyribonucleotide, 2.5 units of RNase
inhibitor, 0.75 mmol/L downstream primer, and 1 U of reverse
transcriptase (Life Technologies). The mixture was incubated at
37 °C for 1 h and at 95 °C for 5 min. The PCR mixture
contained 20 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L
MgCl2, 0.15 mmol/L each primer, 0.2 mmol/L each
deoxyribonucleotide, and 2.5 U of Taq polymerase (Perkin-Elmer Cetus)
in a total volume of 100 µL. The mixture was amplified in a thermal
cycler (Gene Amp PCR System 9600; Perkin-Elmer Cetus) as
follows: 22 cycles of 94 °C for 25 s, 58 °C for 30 s,
and 70 °C for 30 s.
The amplified products (4 µL) were spotted, using a semiautomatic robotic dot-blot procedure (18), on a nitrocellulose filter (Hybond N+; Amersham). The filter was hybridized with a specific oligoprobe labeled with 32P at the 3' end by the T4 polynucleotide kinase (Biolabs), which has the following sequence: 5'-GGCAGGGACAAATGCCGAG-3'. The hybridization conditions were as follows: 65 °C for 1 h, after which the temperature was left to decrease (~8 h) to 25 °C. The filter was washed for 5 min at room temperature with 5x standard saline citrate (1x = 0.15 mol/L NaCl, 0.015 mol/L sodium citrate), and then at 37 °C for 15 min with 2x standard saline citrate.
The number of amplified DNA copies was calculated by measuring the radioactivity of the labeled oligoprobe annealed, using a radioanalytic imaging system (Molecular ImagerTM; Bio-Rad). The radioactivity was expressed as pixel density units. The amount of amplified DNA was calculated for each sample by interpolating the result as pixel density units on a log-log chart, using a calibration curve generated with the scalar dilutions of the RNA calibrator. The amount of the specific mRNA is thus reported as amol/750 ng of the total RNA.
The optimal reverse transcription conditions had been evaluated previously on three calibrators at different concentrations, using 20 cycles of PCR amplification. We evaluated three different temperatures (37, 42, and 45 °C) and three different times (30, 45, and 60 min). The best result (in terms of linearity) was obtained at 37 °C for 60 min. To determine the limiting number of PCR amplification cycles, all of the scalar dilutions of the mRNA calibrator (0.03–300 amol) were amplified at a decreasing number of cycles starting from 30. The efficiency of PCR amplification was constant up to 24 cycles.
imprecision of the method
To evaluate the within-day imprecision of the assay, we
analyzed eight times, in the same experimental series and with the same
calibration curve, three RNA samples containing different amounts of
aldolase A mRNA. Similarly, we tested the between-day imprecision of
the method on the same three samples in six different experiments with
six calibration curves constructed at the same time.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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shows the linearity of the assay obtained after an reverse
transcription reaction for 60 min at 37 °C, and after 24 PCR cycles
using the aldolase A mRNA calibration solution at different dilution
points. The lower detection limit of the method was 0.03 amol, and the
assay was linear up to 300 amol. The range of linearity was very
dynamic, so that specific mRNAs could be measured in different
biological samples in amounts ranging over a difference of five orders
of magnitude (see Fig. 1
). The overall analytical sensitivity of our
procedure was comparable to that reported by Pane et al.
(13), and by others who used either competitive
(12) or multistandard (11) RT-PCR procedures. The
procedure described in this study is highly specific because in
addition to the two specific oligoprimers used for the PCR, an
oligonucleotide complementary to the cDNA is also used to reveal and
quantify the amplified product.
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The within- and between-day imprecision of the method as tested on
three samples with different mRNA concentrations is shown in Table 1
. Again, the precision of the procedure was good (CV
<15% in all instances) over a wide range of mRNA
concentrations. The reproducibility of the method is comparable to that
reported by others who used triple-primer PCR (19). In
agreement with the latter study, our study confirms that once
extracted, the RNA must be analyzed by agarose electrophoresis in the
presence of formaldehyde to verify the integrity and purity of the RNA.
Furthermore, because the specific mRNA is quantified with reference to
the total amount of RNA, it is necessary to exclude DNA contamination
from the RNA. This can be done on several RNA samples by PCR
amplification of a gene (in our case, several exons of the cystic
fibrosis gene). Similarly, the time and temperature of the reverse
transcription reaction and the number of PCR cycles must be strictly
controlled. We found that a difference of 5 min or of 2 °C in the
reverse transcription reaction could affect by ~20% the quantitative
mRNA analysis (data not shown). The good reproducibility obtained in
this study also depended on the use of the robotic workstation, which
limited errors and costs in the analysis of large series of samples.
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The use of a calibrator with the same sequence as the target product overcomes the drawback of competitive RT-PCR procedures, i.e., the different sequences and, thus, the potentially different efficiencies of the RT-PCR amplification between the calibrator and the target mRNA (11). The calibrator used in our noncompetitive procedures has the same sequence as the target mRNA, and thus both calibrator and target mRNA are amplified with the same efficiency. In most noncompetitive procedures, however, the calibrator is obtained by cloning, which is a more complex procedure than the in vitro synthesis used in the present study.
In addition to aldolase A mRNA, we quantified a housekeeping mRNA, i.e., ß-actin, in all tissue samples; no significant differences between HCC and the other groups of patients were obtained (Scheffé multiple comparison test). In any case, we referred the concentrations of aldolase A mRNA to total RNA because various factors such as circadian clock, hypoxia, and neoplasia could influence the concentrations of housekeeping mRNAs (20)(21)(22).
aldolase a mRNA in chronic liver diseases
The concentrations of aldolase A mRNA obtained in liver samples
from the three groups of patients bearing chronic liver diseases and in
the healthy controls are shown in Table 2
. Aldolase A mRNA concentrations were significantly higher in
patients with HCC than in the other groups of patients and in healthy
subjects (P <0.01, two-sided Scheffé multiple
comparison test). The aldolase A concentrations in nonneoplastic liver
diseases were comparable to those obtained in healthy subjects.
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We evaluated aldolase A mRNA within the HCC tissue and in the
surrounding cirrhotic tissue in pairs of biopsy samples from the same
patients. Fig. 2
shows an example of the results. The mRNA concentrations were
clearly higher in the HCC cells than the cirrhotic tissue. Fig. 3
shows the ratio of aldolase A mRNA concentrations between HCC
and surrounding cirrhotic tissue in the 12 pairs of biopsy samples; in
all cases, aldolase A mRNA expression was higher in the HCC cells, with
a ratio between the two values ranging from 1.2- to 21.3-fold.
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Our data confirm, on a quantitative basis, the strong resurgence of aldolase A in HCC at the mRNA level. Aldolase A is expressed exclusively by fetal liver (5), not the adult tissue, and is involved in the glycolytic pathway (to split or synthesize fructose 1,6-diphosphate), whereas the adult liver expresses mainly the aldolase B isoenzyme (involved in the metabolism of exogenous fructose). In fact, serum concentrations of aldolase A have been suggested as a diagnostic tool in HCC patients (23), and our group has revealed higher concentrations of total aldolase and aldolase A in serum from HCC patients compared with patients with cirrhosis (unpublished results). The concentrations of total serum aldolase in the patients from the present study were always within the reference intervals, and no significant differences were obtained between the various groups of patients. This could reflect the different stage of HCC patients.
The various mRNA aldolase A species found in human tissues differ in the 5' noncoding region because of alternative splicing (24), and one of these is expressed by human fetal liver cells. The primers used in the present study hybridize the region common to all the aldolase A mRNA species present in human tissues, so that results obtained from different diseases or tissues can be compared.
Because of the altered morphology of the cirrhotic liver, imaging techniques do not always lead to an unequivocal diagnosis of HCC in cases with liver lesions (1), particularly in cirrhotic patients showing nodular lesions, most of which are benign macroregenerative nodules. Similarly, cytohistology could fail to identify objectively the switch from cirrhosis to HCC (2). In this context, the analysis of specific mRNAs on the biopsy sampled for pathology could be objectively contributory, increasing the number of HCC patients who could benefit from liver resection.
We previously reported that a panel of serum biochemical markers has a
high diagnostic efficiency in differentiating between HCC and cirrhosis
(3). Several of these biochemical signals (i.e.,
-fetoprotein, lactate dehydrogenase, and 
-glutamyltranspeptidase
isoenzymes) are specifically produced or overexpressed by neoplastic
cells. Therefore, quantitative analysis of their mRNA species can be
added to the analysis of aldolase A mRNA as a contributory tool for the
diagnosis of HCC.
In conclusion, the noncompetitive RT-PCR procedure for aldolase A mRNA quantitative analysis described here is highly efficient, reproducible, and with small cost, can be extended to the analysis of other mRNA species. In addition, the quantitative analysis of aldolase A mRNA can, together with cytohistologic procedures, play a role in the diagnosis of HCC.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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fetoprotein by isoelectric focusing. Cancer 1994;74:25-29.
[Web of Science][Medline]
[Order article via Infotrieve]
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