Construction of cDNA bank from biopsy specimens for multiple gene analysis of cancer.
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.)  (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). (4) 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[degrees]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.
[FIGURE 1 OMITTED]
Materials and Methods
Cell culture media and serum, PBS, buffer-saturated phenol, reagents for the synthesis of the first strand of the cDNA, rabbit globin mRNA, vanadyl ribonucleoside complex (VRC) (Gibco BRL, Gaithersburg, MD), reagents for the synthesis of the second strand of the cDNA (Intermountain, Bountiful, UT), reagents for PCR and RNA transcription, streptavidin Magnesphere particles (Promega, Madison, WI), [[[alpha]-.sup.32]P]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  was kindly provided by R.R. Reed (Johns Hopkins University, Baltimore, MD). All other chemicals were purchased from Sigma (St. Louis, MO).
Ten patients with colorectal cancer were subjects for this study after informed consent was obtained. In each patient, ~0.5 [cm.sup.3] 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.
The plasmid pGEM-2 (Promega) containing the rat G protein cDNA  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[degrees]C for 1 h. After the reaction mixture was treated with 1 U of RNase-free DNase at 37[degrees]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 Mg[Cl.sub.2]) 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 .
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[degrees]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 [micro]L 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 . In brief, 50 [micro]L 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  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 .
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 Mg[Cl.sub.2]; 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[degrees]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 [[sup.32]P]dCTP and oligo(dT) as a primer, to compare the synthesis of the cDNA between the Gene-Plate and the conventional methods.
Reaction buffer was replaced with 25 mmol/L Tris, pH 7.5, containing 100 mmol/L KCl; 5 mmol/L Mg[Cl.sub.2]; 10 mmol/L [(N[H.sub.4]).sub.2]S[O.sub.4]; 0.15 mmol/L [beta]-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[degrees]C to synthesize the ds cDNA on the plate [1, 4, 5]. The synthesized second strand of the cDNA was removed by adding 50 mL of boiling water for 10 min. In some experiments, dCTP was replaced with [[sup.32]P]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 [T.sub.m] of 55[degrees]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 [beta]-actin (sense: 5'-CTTCGCGGGCGACGATGC-3', antisense: 5'-CGTACATGGCTGGGGTGTTG-3'), 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[degrees]C overnight, dried, resuspended in water at 0.1 g/L, and stored at -20[degrees]C until use.
One to five microliters of the template DNA was mixed with 0.2 [micro]L each of 10 mmol/L dATP, dGTP, dCTP, and dTTP and 0.5 [micro]L each of sense and antisense primers, 0.5 [micro]L of 25 mmol/L Mg[Cl.sub.2], 1 mL of PCR buffer, and 0.1 [micro]L of Taq polymerase (Promega) in a final volume of 10 [micro]L. PCR was carried out in a DNA thermal cycler (Model 480; Perkin-Elmer, Norwalk, CT) with 30 cycles of annealing temperature at 55[degrees]C for 1.5 min, 72[degrees]C extension for 4 min, and 95[degrees]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 [beta]-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 [[sup.32]P]CDNA
[[sup.32]P]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.
SYNTHESIS OF THE FIRST STRAND OF THE CDNA
The cytosolic RNA derived from 5 X [10.sup.5] human mononuclear leukocytes was first applied to either the oligonucleotide-free control plates or the oligonucleotide-immobilized GenePlate for hybridization. After unbound materials were removed by aspiration, some wells were kept dry and others were processed for the synthesis of the cDNA, and the amounts of hybridized mRNA and the synthesized cDNA were quantified by Yoyo-1 simultaneously . If Yoyo-1 was applied to the plates before hybridization, Yoyo-1 fluorescence was significantly higher in the GenePlate than in control plates (Fig. 2, blank). This confirmed our previous publication , 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 .
[FIGURE 2 OMITTED]
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).sup.+] mRNA was purified from mouse liver as previously described , and the first and second strands of the cDNA were synthesized either in solution or on the GenePlate in the presence of [[sup.32]P]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 [[sup.32]P]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.
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.
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
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 [cm.sup.3] 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 [beta]-actin gene. As shown in Fig. 5, the [beta]-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[degrees]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).
[FIGURE 5 OMITTED]
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 [beta]-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 [beta]-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).
Many genes and gene products still require analysis, especially in tumors. For example, cytokines and their specific receptors, adhesion molecules and receptors, oncogenes, transcription factors, cyclins, growth-promoting enzymes, proteases/peptidases, growth and angiogenesis factors, and apoptosis-related genes have been shown to play important roles in cancers. Although basic research provides detailed analysis for each gene by using a relatively simple model with artificial environments, medical researchers need to analyze multiple genes in clinical isolates from cancer patients with various stages of disease that are either treated or untreated. Although various biotechnology tools are available for the analysis of genes and gene function, it is still not easy to analyze small clinical specimens. Therefore, our main focus in the present study was to develop a new research system, the cDNA bank, that is useful for multiple gene analysis from single small clinical specimens.
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  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 . 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  is much more sensitive and a less labor-intensive method for the analysis of mRNA compared with conventional Northern blotting , RT-PCR is used frequently in clinical research . 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 , 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 . 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.
We thank R.R. Reed (Johns Hopkins Univ.) for providing us the G protein cDNA. We also thank T. Chishima, H. Yamaoka, H. Katamura, K. Matsuo, S. Imai, and S. Ooki (Dept. of the Second Surgery, Yokohama City Univ., Yokohama, Japan) for their support of obtaining clinical specimens, K. Fujimoto, T. Hosokawa, H. Ohno, M. Yamaki (Hitachi Chemical, Japan), Rici de Fries, Mieko Ogura (Hitachi Chemical Research Center), Y. Tsukada, K. Hiromura, H. Mitsuhashi (Dept. of the Third Internal Medicine, Gunma Univ., Japan), I. Kusumi, T. Ishikane (Dept. of Psychiatry, Hokkaido Univ., Japan), T. Shinagawa (Dept. of the Third Internal Medicine, Nagasaki Univ., Japan), K. Tominaga (Dept. of the Third Internal Medicine, Osaka City Univ., Japan), A.S. Tarnawski, and I.J. Sarfeh (DVA Medical Center, Long Beach) for their contribution in our preliminary studies, and Steve Disper (Hitachi Chemical Research Center) for synthesizing oligonucleotides. This study was supported by a grant from Hitachi Chemical Research Center; a Grant-in-Aid for Scientific Research, no. 22701; the Ministry of Education, Science, Sports, and Culture, Japan; and the 15th grant from the Japanese Foundation for Multidisciplinary Treatment of Cancer.
Received August 29, 1996; revised December 17, 1996; accepted December 17, 1996.
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TAKASHI ISHIKAWA, (1) YASUSHI ICHIKAWA, (1) YASUHIKO MIURA, (1) MOBUYOSHI MOMIYAMA, (1) CYLIA KELLER, (2) KENNETH KOO, (2) TATSUO AKITAYA, (3) HIROSHI SHIMADA, (1) and MASATO MITSUHASHI (2) *
(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.
(4) Nonstandard abbreviations: ds, double stranded; ODC, ornithine decarboxylase; VRC, vanadyl ribonucleoside complex; DEPC, diethylpyrocarbonate; DTT, dithiothreitol; RT, reverse transcription; and ss, single stranded.
* Author for correspondence. Fax 714-725-2727; e-mail HCN02644@niftyserve.or.jp.
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|Title Annotation:||Molecular Pathology|
|Author:||Ishikawa, Takashi; Ichikawa, Yasushi; Miura, Yasuhiko; Momiyama, Mobuyoshi; Keller, Cylia; Koo, Kenn|
|Date:||May 1, 1997|
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