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Articles |
1
The Second Department of Surgery, Yokohama City University, School of Medicine, Yokohama, Japan.
2
Hitachi Chemical Research Center, 1003 Health
Sciences Rd. West, Irvine, CA 92612.
3
Hitachi Chemical Co., Ltd., Ibaraki, Japan.
a Author for correspondence. Fax 714-725-2727; e-mail HCN02644{at}niftyserve.or.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: mRNA • reverse transcription–polymerase chain reaction • oligonucleotides
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous studies, we developed a unique assay system in which
mRNA is specifically captured on plastic plates by the poly(dT)
sequence of immobilized oligonucleotides (Fig. 1
, step I–II), followed by synthesis of the first (Fig. 1
, step
III) and the second strand of cDNA on the plate (GenePlate; Hitachi
Chemical Research Center and Hitachi Chemical Co.) (1)
(Fig. 1
, step IV). Once the double-stranded (ds) cDNA is formed on the
plate, the sense strand of the cDNA can be easily dissociated from the
plate and is used as a template for gene amplification procedures to
detect or quantify expression of various genes (Fig. 1
, step
V).1
In the present study, we have successfully
constructed a "cDNA bank" on the GenePlate from needle biopsy-size
specimens of colorectal cancers and surrounding normal colon mucosa
from the same individuals, and determined whether this system is
applicable to clinical oncology research. We report here that cDNA was
repeatedly synthesized on the plate in good quality even after
long-term storage at 4 °C for 6 months. After conducting PCR
amplification of these cancer cDNAs, we also found that
cancer-associated genes such as the ornithine decarboxylase (ODC) gene
were expressed differently in cancers compared with surrounding normal
mucosa from the same individuals. Therefore, the proposed cDNA bank may
provide unique tools for clinical oncologists to analyze various genes
from a single clinical specimen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]dCTP (29.6 TBq/mmol) (DuPont, Boston, MA),
IsoLymph (Gallard-Schlesinger, Carle Place, NY), Yoyo-1 (Molecular
Probes, Eugene, OR), chloroform, isoamyl alcohol (Fisher, Tustin, CA),
and GenePlate (Hitachi Chemical Research Center, Irvine, CA, and
Hitachi Chemical Co., Ibaraki, Japan) were supplied from the designated
suppliers. The rat G protein cDNA (2) was kindly provided
by R.R. Reed (Johns Hopkins University, Baltimore, MD). All other
chemicals were purchased from Sigma (St. Louis, MO).
patients
Ten patients with colorectal cancer were subjects for this study
after informed consent was obtained. In each patient, ~0.5
cm3 of both tumor and surrounding normal tissue were
separately removed from either needle biopsy or surgical specimens, and
were instantly frozen and kept in liquid nitrogen until use. Tumors
were examined histologically to confirm the presence of cancer cells.
Normal tissues were also examined histologically to confirm that the
regions were cancer-free.
mrna synthesis
The plasmid pGEM-2 (Promega) containing the rat G protein cDNA
(2) was linearized with NheI, then purified
with two rounds of ethanol precipitation to remove contaminating
RNases. The mRNA was synthesized with T7 RNA polymerase at 37 °C for
1 h. After the reaction mixture was treated with 1 U of RNase-free
DNase at 37 °C for an additional hour, synthesized mRNA was purified
with one round of phenol extraction followed by ethanol precipitation.
The quality of mRNA was determined by electrophoresis and stained with
ethidium bromide in 1% agarose gel.
extraction of nucleic acid
Heparinized blood taken from healthy adults was diluted threefold
with PBS and layered onto IsoLymph. After centrifugation at
400g for 30 min at room temperature, the interphase
containing mononuclear leukocytes was removed and washed with PBS three
times. Cells were resuspended in lysis buffer (50 mmol/L Tris, pH 8.0,
0.5 mol/L NaCl, 5 mmol/L MgCl2) containing 5 mL/L NP-40 and
20 mmol/L VRC, and incubated on ice for 5 min. After centrifugation at
10 000g for 2 min to precipitate genomic DNA and cell
debris, supernatant solutions were applied to the GenePlate for
hybridization as previously described (3).
To each frozen tissue sample, 2–3 mL each of extraction buffer (50 mmol/L Tris, pH 7.6, 5 mmol/L EDTA, 0.05 mol/L NaCl, 5 g/L sodium dodecyl sulfate) and buffer-saturated phenol (phenol:chloroform:isoamyl alcohol 25:24:1) was added, then immediately homogenized with a Polytron (Kinematica, Littav, Switzerland). After centrifugation at 8000g at 4 °C for 5 min, supernatant solutions were transferred to fresh tubes, and phenol extraction was repeated for an additional one to three times until no debris was found in the interphase, followed by ethanol precipitation. After washing with 750 mL/L ethanol, dried nucleic acids were suspended in 200 µL of diethylpyrocarbonate (DEPC)-treated hybridization buffer (10 mmol/L Tris, pH 7.5, 1 mmol/L EDTA, 0.5 mol/L NaCl), and applied to the GenePlate for hybridization (4)(5).
fluorometric measurement of mrna
The total amount of mRNA was determined by our method
(3). In brief, 50 µL each of nucleic acid samples was
applied to wells of the GenePlate, and incubated at room temperature
for 1 h to allow mRNA to hybridize to the dT sequence of the
immobilized oligonucleotides on the plate. Unbound materials were
removed by aspiration and washed with low-salt buffer (10 mmol/L Tris,
pH 7.6, 1 mmol/L EDTA, 0.1 mol/L NaCl) twice. Fifty microliters of
Yoyo-1 (6) in a final dilution of 1:1000 was added to each
well, and the fluorescence intensity of each well was measured by a
fluorescent plate reader (CytoFluor 2300 and 2350; Millipore, Bedford,
MA) with excitation and emission wavelengths of 485 nm (bandwidth 20
nm) and 530 nm (bandwidth 25 nm), respectively
(7)(8). The amount of mRNA in test samples was
determined by comparing their Yoyo-1 fluorescence to that of the known
concentrations of the rabbit globin mRNA as a calibrator
(3).
cdna synthesis
Tissue extracts containing ~200 ng of mRNA were applied to the
fresh GenePlate for hybridization. After a 1-h incubation at room
temperature as described above, the plate was washed with the low-salt
washing buffer twice, and the first strand of the cDNA was synthesized
on the plate by replacing buffer with 50 mmol/L Tris, pH 8.3,
containing 75 mmol/L KCl; 3 mmol/L MgCl2; 10 mmol/L
dithiothreitol (DTT); 10 mmol/L each of dATP, dGTP, dCTP, and dTTP; and
100 U of Moloney murine leukemia virus (MMLV) reverse transcriptase
(Gibco BRL) at 37 °C for 1 h
(1)(4)(5). In some experiments,
Yoyo-1 was added to each well to quantify the amount of freshly
synthesized cDNA on the plate. In parallel experiments, the sense
strand of the cDNA was also synthesized in solution in the presence of
[32P]dCTP and oligo(dT) as a primer, to compare the
synthesis of the cDNA between the GenePlate and the conventional
methods.
Reaction buffer was replaced with 25 mmol/L Tris, pH 7.5, containing 100 mmol/L KCl; 5 mmol/L MgCl2; 10 mmol/L (NH4)2SO4; 0.15 mmol/L ß-NAD; 250 mol/L each of dATP, dGTP, dCTP, and dTTP; 1.2 mmol/L DTT; 65 kU/L DNA ligase; 250 kU/L DNA polymerase; and 13 kU/L RNase H (Intermountain), and incubated overnight at 16 °C to synthesize the ds cDNA on the plate (1)(4)(5). The synthesized second strand of the cDNA was removed by adding 50 µL of boiling water for 10 min. In some experiments, dCTP was replaced with [32P]dCTP during the ds cDNA synthesis in both the GenePlate and the conventional solution assays.
primer sequence design and synthesis
Primer sequences were determined by using the computer program
HYBsimulator (9)(10) and appropriate design
strategy (11)(12). In brief, oligonucleotide
sequences with Tm of 55 °C were extracted
from every position of the target gene of interest, each
oligonucleotide sequence was screened for possible cross-hybridizable
genes, and their binding strength against gene sequences registered in
GenBank. Primer sequences for the ß-actin (sense:
5'-CTTCGCGGGCGACGATGC-3', antisense: 5'-CGTACATGGCTGGGGTGTTG3'), the G
protein (sense: 5'-GCCAACAAAAAGATCGAGAAGC-3', antisense:
5'-CATGTGGAAGTTGACTTTGTCC-3'), ODC (sense:
5'-GACTCTGGAGTGAGAATCATA-3', antisense: 5'-ATCCAATCACCCACATGCATT-3'),
and the jun (sense: 5'-CCCTGAAGGAGGAGCCGCAGAC-3',
antisense: 5'-CGTGGGTCAAGACTTTCTGCTTGAGCTG-3')
(4)(5) primer sequences were the most specific
ones with minimum chance of cross-hybridization against other unrelated
gene sequences in primate and rodent databases in GenBank.
Resulting oligonucleotides were synthesized by the DNA synthesizer 380 B type (Applied Biosystems, San Jose, CA), treated with ammonium hydroxide at 55 °C overnight, dried, resuspended in water at 0.1 g/L, and stored at -20 °C until use.
pcr
One to five microliters of the template DNA was mixed with 0.2
µL each of 10 mmol/L dATP, dGTP, dCTP, and dTTP and 0.5 µL each of
sense and antisense primers, 0.5 µL of 25 mmol/L MgCl2, 1
µL of PCR buffer, and 0.1 µL of Taq polymerase (Promega) in a final
volume of 10 µL. PCR was carried out in a DNA thermal cycler (Model
480; Perkin-Elmer, Norwalk, CT) with 30 cycles of annealing temperature
at 55 °C for 1.5 min, 72 °C extension for 4 min, and 95 °C
denaturation for 1.5 min, as previously described
(4)(5). After PCR, amplified genes were
analyzed by agarose gel electrophoresis followed by staining with
ethidium bromide. The expected sizes of PCR products of ß-actin, ODC,
and jun were 322, 347, and 189 bp respectively. These PCR
products were further confirmed by the appropriate restriction enzyme
digestions (data not shown).
agarose gel electrophoresis of [32p]cdna
[32P]cDNAs synthesized on the plate or in solution
were separated by electrophoresis in 1% agarose gel. After
electrophoresis, the gel was wrapped and exposed to x-ray films with an
intensifying screen at room temperature for 3 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, blank). This confirmed our previous publication
(7), in which the amount of immobilized oligonucleotides
was quantified by Yoyo-1. After RNA hybridization, Yoyo-1 signals
increased in both plates (Fig. 2
, mRNA). However, after adding hot
water to dehybridize mRNA and removing mRNA by aspiration, Yoyo-1
fluorescence was reversed only in the GenePlate, but not in the control
plates (Fig. 2
, de-hyb.). Therefore, this reversible portion of Yoyo-1
fluorescence indicates the amount of hybridized mRNA on the plate, as
previously described (3).
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Because our preliminary experiments suggested that the sensitivity of
Yoyo-1 fluorescence of single-stranded (ss) DNA is identical to that of
ss RNA (data not shown), the synthesized cDNA was denatured to remove
template mRNA before Yoyo-1 analysis. Yoyo-1 fluorescence of the
synthesized ss cDNA was similar to that of hybridized mRNA (Fig. 2
, cDNA). This Yoyo-1 method is ideal for monitoring the synthesis of the
first strand of the cDNA because of its simplicity, rapidity, and
nonradioactive procedure. Therefore, this test was repeated many times
as part of quality-assurance protocol of the proposed cDNA bank.
synthesis of the second strand of the cdna
To analyze the size distribution of the second strands of the
synthesized cDNA, poly(A)+ mRNA was purified from mouse
liver as previously described (4), and the first and
second strands of the cDNA were synthesized either in solution or on
the GenePlate in the presence of [32P]dCTP. As shown in
Fig. 3
, the synthesized second strand of the cDNA appears on the plate
as a smear (lane 3), which was approximately equivalent to that of the
first strand of the cDNA synthesized in solution (lane 1), whereas the
second strand of the cDNA synthesized in solution failed to show this
similar smear pattern (i.e., less high-molecular-mass material) (lane
2). Furthermore, the second strand of the cDNA was repeatedly
synthesized, in the presence of [32P]dCTP, from the same
plate used once for the ds cDNA synthesis. Interestingly, the quality
of the second-round synthesis of the sense strand of the cDNA (lane 4)
was equivalent to that of the first-round synthesis of the antisense
strand (lane 1) and the sense strand of the cDNA (lane 3), although the
cDNA synthesis reaction did not contain random hexamer primers.
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In another experiment, various concentrations of the in vitro
synthesized rat G protein mRNA were applied to the GenePlate for
hybridization, followed by the synthesis of the first and second
strands of the cDNA. The second strand of the cDNA was recovered from
the plate by heat denaturation, after which two rounds of cDNA
synthesis were performed on the same plate without random hexamer
primers. The recovered cDNA was used for PCR to amplify the G protein
gene product. As shown in Fig. 4
, the G protein cDNA was amplified from 1–10 pg of mRNA in a
dose-dependent manner, even after second or third rounds of cDNA
synthesis on the same plate.
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The sensitivity of this assay is dependent on the type of mRNA, buffer, presence of RNase inhibitors, and sequences of PCR primers. For example, when commercially available rabbit globin mRNA was applied to the plate in the presence of 10 mmol/L VRC, PCR products were seen after agarose gel electrophoresis from 100 fg of mRNA (data not shown). Furthermore, PCR products were seen even after the 10th round of cDNA synthesis on the same plate (manuscript in preparation).
cdna synthesis from clinical materials
We first determined the optimal lysis procedures and the amount of
tissue necessary for the GenePlate experiments. After many
trial-and-error experiments, the direct phenol extraction procedure as
described in Materials and Methods was determined to be the
fastest and the most efficient and reproducible method for solid
tissues. Furthermore, samples as small as needle biopsy specimens of
~0.5 cm3 from human stomach, colon, heart, and kidney
were found to be sufficient for at least triplicate wells of the
GenePlate, although amount of recovered mRNA varied widely among type
of tissue (data not shown).
In the present study, total nucleic acids were prepared from colorectal
tumor and surrounding normal mucosa, and applied to the plate for
hybridization before the synthesis of the first and second strands of
the cDNA. The second strand of the cDNA was removed from the plate,
then used as a template for PCR to amplify cancer-related genes such as
the ODC and the jun gene as well as the control ß-actin
gene. As shown in Fig. 5
, the ß-actin (1a), ODC (2a), and jun (3a) were
seen on agarose gels in all 10 clinical samples in both tumor (T) and
normal tissues (N). All cDNAs were of the expected sizes. Furthermore,
PCR products were also seen in the cDNA synthesized from the once-used
GenePlate stored at 4 °C for 6 months (Fig. 5
1b, 2b, and 3b).
Interestingly, the intensity of PCR products from the second-round
synthesis of the cDNA was similar to that of the first-round cDNA
synthesis (Fig. 5
).
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Because the same amount of total mRNA was applied to the plate, and the
cDNA synthesis was conducted simultaneously, the resulting PCR products
of the control ß-actin gene were similar among different individuals
and especially between tumor and normal mucosa (Fig. 5
). According to
the results of Fig. 4
, the intensity of PCR products was correlated
with the amount of applied specific mRNA. Therefore, variation of the
intensity of PCR products among different individuals may be due to the
difference of the fraction of specific mRNA per total mRNA.
More interestingly, PCR products of the ß-actin and the
jun oncogenes appeared with almost the same densities
between tumor and normal mucosa within the same individuals, whereas
PCR products of the ODC were significantly more in tumor than
accompanying normal mucosa (Fig. 5
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the most interesting results in this study was the synthesis of
the cDNA in a primer-independent fashion on the GenePlate (Figs. 3
, 4
, and 5
). The method for the first-round synthesis of the ds cDNA was the
same as that of a standard protocol (13) in which the RNA
strand of the mRNA-cDNA complex is digested with RNase H, and the
second strand of the cDNA is believed to be initiated by partially
digested mRNA as primers. However, during the second- and third-round
syntheses of the ds cDNA as shown in Figs. 3
, 4
, and 5
in the present
study, the second strand of the cDNA was successfully synthesized
without primers or any mRNA fragments. Furthermore, the quality (Fig. 3
) and sensitivity (Figs. 4
, 5
) of the second- and third-round
syntheses of the cDNA was similar to those of the first-round synthesis
of the cDNA. If the 3' end of the first strand of the cDNA folds back
and acts as a primer for the synthesis of the second strand of the
cDNA, the second strand of the cDNA should not be removed from the
plate without using S1 nuclease or equivalent (14).
However, in our experiments, the second strand of the cDNA was easily
removed by heat denaturation alone. If some ss DNA or its fragments are
present in the applied materials, and are nonspecifically bound to the
plate, they may act as primers during second-strand synthesis. However,
because such DNA and DNA-primed cDNA are dissociated during the heat
denaturation step at the end of the first round of cDNA synthesis, they
are no longer available for second and third rounds of cDNA synthesis.
Although we do not know the exact mechanism of primer-independent cDNA
synthesis on the plate, this phenomenon was always reproducible, even
with different collaborators.
To analyze whether primer-independent synthesis of the second strand of the cDNA is a unique event on the GenePlate or not, human leukocyte mRNA was also hybridized with biotinylated oligo(dT), followed by reaction with streptavidin-coupled magnetic particles (Magnesphere, Promega). The first- and second-round syntheses of the ds cDNA were conducted on magnetic particles with or without random hexamer primers, and the synthesized sense strand of the cDNA was used for PCR to amplify various genes. As a result, the PCR products from the reaction with random hexamer primers were not significantly different from that of primer-independent reactions (data not shown). Therefore, we believe that primers may not be required for the second strand of the cDNA synthesis on solid surfaces.
Because reverse transcription (RT)-PCR (15) is much more
sensitive and a less labor-intensive method for the analysis of mRNA
compared with conventional Northern blotting (16), RT-PCR
is used frequently in clinical research (15). However, a
major drawback of RT-PCR is the time-consuming step of the purification
of mRNA/total RNA from clinical specimens, and the difficulty of
quantification. Although recent quantitative PCR technology provides
quantitative results of PCR or RT-PCR (17),
standardization is also essential to compare gene expression among
various tissues in different individuals. For example, the dry weight
or wet weight of samples can be used to standardize the data of gene
expression among different clinical specimens; however, tumors often
contain debris and (or) necrotic tissues. The number of living cells is
difficult to obtain from solid tumors. If the same amount of total
nucleic acids or total RNA is used for comparison, the interpretation
of the results is difficult because the amount of mRNA may vary widely
among tested materials. Therefore, in the present study, an equal
amount of mRNA was applied to the GenePlate for cDNA synthesis,
followed by PCR. As a result, the difference of ODC gene expression
between tumors and normal tissues was observed on simple agarose gels
subjected to electrophoresis and stained with ethidium bromide (Fig. 5
). The result of the ODC mRNA expression is consistent with the
previous report of the ODC enzyme assay in colon cancer
(18). Because of the dose dependency of the intensity of
PCR products on agarose gels (Fig. 4
), the difference of intensity of
PCR products in Fig. 5
may reflect the amount of specific mRNA in test
materials.
The cDNA bank constructed from the same amount of starting mRNA from clinical specimens provides a useful tool for clinical oncology research to screen expression of various genes among different cancers. Because of its easily manipulated format of 96-well microtiter plates, rapid quality-assurance procedure for the amount of immobilized oligonucleotides and hybridized mRNA by Yoyo-1 fluorescence, and capability of long-term storage and multiple reproduction of the cDNA, we believe that the cDNA bank may become a common method to provide standardized cDNA materials to researchers in the future. These standardized cDNA may be used not only for individual gene analysis, but also for application to differential mRNA display for new gene discovery and mRNA fingerprinting.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  M. Mitsuhashi, S. Tomozawa, K. Endo, and A. Shinagawa Quantification of mRNA in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA Synthesis Clin. Chem., April 1, 2006; 52(4): 634 - 642. [Abstract] [Full Text] [PDF]
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Y. Hamaguchi, Y. Aso, H. Shimada, and M. Mitsuhashi Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates Clin. Chem., November 1, 1998; 44(11): 2256 - 2263. [Abstract] [Full Text] [PDF]
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K. Eguchi, Y. Hamaguchi, Y. Aso, T. Shioiri, M. Ogura, and M. Mitsuhashi Oligo(dT)-immobilized Pipette Tip: Efficient New Methodology for mRNA Preparation and Direct Gene Amplification Clin. Chem., October 1, 1998; 44(10): 2208 - 2210. [Full Text] [PDF]
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) or the control plates (
), in which no
oligonucleotide was immobilized. Hybridization buffer alone was also
applied to plates as control (blank). After a 1-h incubation at room
temperature to capture mRNA (mRNA), some wells were washed with 50 µL
of boiling DEPC-water twice to dissociate hybridized mRNA (de-hyb.).
The first strand of the cDNA was then synthesized on the plate, as
described in the text. After the cDNA synthesis, the template mRNA was
removed by addition of boiling water (cDNA). Fifty microliters of
Yoyo-1 in a final dilution of 1:1000 was added to each well, and the
fluorescence intensity of each well was measured simultaneously in a
fluorescent plate reader as described in the text. All data were the
mean ± SE from triplicate determinations. This experiment was
reproduced at the time of manufacturing of the GenePlate as a
quality-assurance protocol.
