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13.1 Introduction .203. 13.2 Materials and Methods .204. 13.2.1 Primer Design. 204. 13.2.2 RNA Isolation and Reverse Transcription. 204. 13.2.3 PCR. 205.
LUMINESCENCE BIOTECHNOLOGY Instruments and Applications

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Differential Display Polymerase Chain Reaction Using Chemiluminescent Detection Gang An and Robert W Veltri

CONTENTS 13.1 Introduction 13.2 Materials and Methods 13.2.1 Primer Design 13.2.2 RNA Isolation and Reverse Transcription 13.2.3 PCR 13.2.4 Electrophoresis and Chemiluminescent Detection 13.2.5 Reamplification, Sequencing, and Confirmation of Expression 13.3 Results and Discussion Acknowledgments References

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13.1 INTRODUCTION Regulation of gene expression plays a fundamental role in many normal and abnormal cellular physiological processes, including normal cell differentiation and development, carcinogenesis, and host responses to many environmental insults and pathological changes. Identification of genes that are differentially expressed in different cell types or under various conditions is of great importance in modem biology. Traditionally, differential hybridization and subtractive hybridization have been used widely to identify genes that express differentially between different but closely related types of cells or tissues.1,2Although many genes have been successfully cloned by these methods, both approaches are rather labor-intensive and time-consuming, and allow simultaneous comparison of only two RNA populations. The recently described differential display polymerase chain reaction (DD-PCR) method3.4have gained popularity in the biomedical research community. The method allows for rapid and relatively complete survey of differential gene expression among various cell or tissue types. In the DD method, mRNAs from two or more cell types are divided into subpopulations and reverse-transcribed into cDNAs using 3'-anchored oligo(dT) primers. cDNAs from different cell types are then amplified by PCR using the same set of 3'-anchored primers in combination with a 5' arbitrary primer. The radioactive cDNA products from PCR are resolved on a DNA sequencing gel in sequentially arranged lanes to allow rapid comparisons. Differentially expressed mRNA species are identified by comparing the autoradiographic intensities of cDNA bands on the gel. The identifiedcDNAs, which were significantlyaltered in their expression, are then reamplified and finally cloned into plasmid. Since the introduction of the DD-PCR method by 0-8493-0719-81021$0.00+$1.50 102002 by CRC Press LLC

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Liang and Pardee,3 other similar methods utilizing different primer design strategies have been described, including RNA fingerprinting using arbitrarily primed PCR (RAP-PCRi and palindromic primer-based DD-PCR.6 Compared with DD-PCR, RAP-PCR uses arbitrary primers in both reverse transcription and PCR amplification, and palindromic primer-based DD-PCR uses only one arbitrary primer in PCR reactions. The majority of the described methods use radioisotopes to detect band patterns on sequencing gels,H which has the technical disadvantages of handling hazardous radioactive material, potential contamination of the working area and thermal cycler, and the cost associated with Nuclear Regulatory Commission licensing as well as those costs related to waste disposal of the radioactive materials. For those who prefer non-isotopic approaches, there are some alternative methods from which to choose. For example, non-isotopic DD-PCR methods have been developed that use silver staining detection,? fluorescent detection,S or chemiluminescent detection.9 Although both the silver staining and the fluorescent labeling DD-PCR methods are valuable alternatives to radioactive DDPCR, the silver staining method is less sensitive and displays much fewer bands than radioisotopicbased method, and the fluorescent labeling method requires an expensive automatic sequencing machine and software. We describe here a DD-PCR method employing the chemiluminescent detection method, which has all the benefits of a non-isotopic method without sacrificing the sensitivity of radioactive detection, and involves no specialized instrumentation. The method described uses four different 5' biotinylated oligo(dT)-anchored primers to reverse-transcribe RNAs from different cell types into four pools of cDNAs. Each cDNA pool is then amplified by PCR using the same set of 5' biotinylated oligo(dT)-anchored primer as in the reverse transcription, combining with an arbitrary primer. The PCR products are separated by standard denaturing gel electrophoresis. About 20% of the DNA fragments is then transferred to a nylon membrane by the capillary method. Following transfer, the DNA fragments are detected by SEQ_Light™ Chemiluminescent DNA Sequencing Detection System (Applied Biosystems, Foster City, CA). The DD-PCR band patterns are obtained by exposing the membrane to standard X-ray film. Exposure times are generally 30 to 60 min. After positive bands are identified, the X-ray film and the nylon membrane are used to locate the band position back to the gel. The remaining DNA (about 80%) on the gel in the identified position is recovered and used for reamplification and cloning. We have successfully used this method to clone genes differentially expressed in prostate cancer.

13.2 13.2.1

MATERIALS AND METHODS PRIMER DESIGN

The four biotinylated oligo(dT) anchored primers (T12VN)are designed as follows where V represents mixed bases of A, G, and C: 5'-biotin-l TIll TTTTTTTVG-3' 5' -biotin- TTTTTTTTTTTTV

A-3'

5'-biotin-TTTTTTTTTTTTVT-3' 5'-biotin-TTTTTTTTTTTTVC-3' The arbitrary decamers can be designed by randomly picking ten bases with about 60% GC content. Differentarbitrary decamerkits are also availablecommercially(OperonTechnologies,Alameda, CA). 13.2.2

RNA ISOLATIONAND REVERSE TRANSCRIPTION

Total RNA is isolated from normal and prostate cancer specimens by the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction methodlOor by TRIZOL reagent (GIBCO/BRL, Gaithersburg, MD) following manufacturer's directions. RNA (10 /lg) from each tissue is treated

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with 5 units of RNase-free DNase I (GIBCOIBRL, Gaithersburg, MD) in the presence of 20 mM Tris-HCI, pH 8.4, 50 mM KCI, 2 mM MgCI2, and 20 units of RNase inhibitor (Boehringer Mannheim, Indianapolis, IN). After extraction with phenoVchloroform and ethanol precipitation, the RNA is redissolved in DEPC-treated H20. Four pools of cDNA are generated using the four different biotinylated oligo(dT)-anchored primers. For each pool, five)1gof the DNase-treated RNA from each tissue is reverse-transcribed into cDNA using one of the biotinylated oligo(dT)-anchored primer and M-MLV reverse transcriptase (GIBCOIBRL) in a total of 40 )11reaction. The reaction mixture contains 50 mM Tris-HCI, pH 8.3, 75 mM KCI, 3 mM MgCI2, 10 mM DTT, 500 J1MdNTP, 2 J1Mbiotinylated oligo(dT)-anchored primer, and 400 U M-MLV reverse transcriptase. The reactions are incubated at room temperature (22°C) for 10 min, followed by a 50-min incubation at 42°e. A final 10-min incubation at 70°C is performed to inactivate the enzyme.

13.2.3

PCR

For each pool of cDNA from differentcells or tissues(e.g., normaland cancer), PCR is performedusing the same biotinylatedoligo(dT)-anchoredprimeras in reversetranscription,combiningwith an arbitrary 10mer.Differentprimer combinationscan be used for different PCR reactionsto generate distinct band patterns. The PCR mixture contains 2)11of cDNA, lO mM (Tris)-HCI,pH 9.3, 50 mM KCI, 1.5 mM MgCI2, 50 J1MdNTPs, 1.25 U of Taq DNA polymerase, and 0.2 J1Meach of the biotinylated oligo(dT)-anchored primer and arbitrary lOmer in a total of 20 )11reaction. The amplification parameters include 40 cycles of reaction with 30 s denaturing at 94°C, 2 min annealing at 40°C, and I min extension at n°e. A final extension at noc is performed for 15 min. After PCR, 12)11 of loading dye (95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol) is added to each tube. 13.2.4

ELECTROPHORESIS AND CHEMILUMINESCENT DETECTION

A 6% denaturing polyacrylamide DNA sequencing gel is prepared following established procedure.) The gel is prerun for about I h to warm up to about 50°e. The PCR products with loading dye are heated at 80°C for 3 min, and 5 )11of each is loaded immediately onto the gel. The gel is run at about 120 W constant power for about 2 h or until the xylene cyanol dye reaches about 20 cm from the bottom. After electrophoresis, DNA fragments on the gel are transferred to the Tropilon-PlusTM nylon membrane (Tropix), or other positive-charged nylon membrane by capillary transfer. Fixing the gels or removing the urea is unnecessary. Approximately 20% of the DNA is transferred to the membrane, which can be easily detected using chemiluminescent detection procedure. The setup for capillary transfer is illustrated in Figure l3.1 and the procedure is as follows:

l. Disassemble the gel apparatus and separate the glass plates. 2. Lay a piece of Whatman@3MM filter paper on top of the gel, making sure the paper is in good contact with the gel. Remove the gel from the glass plate with the filter paper. Place the paper with gel attached back on the plate with the gel side up. 3. Cut a piece of nylon membrane the same size as the region of the gel to be blotted and wet it thoroughly with TBE. 4. Carefully place the wet membrane on the gel. Remove any air bubbles by rolling a pipette over the membrane. 5. Place three pieces of dry Whatman filter paper on top of the membrane, another glass plate, and a 2-kg weight on top. Allow the transfer to proceed for I h. Following the transfer step, orient the membrane and the gel by punching through using a needle with India ink in three locations. The membrane is then carefully separated from the gel. The transferred DNA is immobilized on the membrane by UV irradiation (total exposure: 120 mJoules), or baking at 80°C for I h.

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Filter Paper

Nylon Membrane Glass Plates FIGURE 13.1 Illustration of the capillary transfer method.

The SEQ_Light™ chemiluminescent detection system (Applied Biosystems) is used to detect bands on the sequencing gel following manufacturer's instruction. After detection, DNA band patterns are captured on X-ray film, and differential expressed bands are identified. 13.2.5

REAMPLIFICATlON, SEQUENCING,AND CONFIRMATIONOF EXPRESSION

After positive bands are identified, the X-ray film and the nylon membrane are then used to locate the band position back to the gel. The remaining DNA (about 80%) on the gel in the identified position is recovered. Reamplification is performed following established procedure.3.4 Briefly, each gel slice is soaked in 100 ).ll of H20 in a 1.5-ml centrifuge tube and boiled for 10 min. After centrifuging for 2 min, the supernatant is transferred to a new tube. The DNA is precipitated by adding 10 ).ll of 3 M NaOAc (pH 5.2), 2 ).ll of glycogen (20 mglml), and 250 ).ll of ethanol, followed by incubating at dry ice or -80°C freezer for 20 min and then spinning for 10 min. The pellet is washed with 500 ).ll of 70% ethanol, dried, and resuspended in 10 ).llof H20. Reamplification is performed using 4 ).ll of the DNA and the same set of primers and PCR conditions as in the display experiment. The reamplified DNA band can be used directly as a probe for Northern confirmation or a template for DNA sequencing. Since all the antisense DNA molecule in the reamplified product has a biotin in its 5' end, the reamplified DNA can be used directly as probe for non-isotopic Northern hybridization to confirm the differential expression of the gene. The reamplified band is purified by Qiaex kit (Qiagen, Chatsworth, CA) and eluted into 20 ).ll of H20. Northern hybridization is performed using established procedure.l Then, 10 ).ll of the purified DNA is boiled for 5 min, cooled on ice, and added to hybridization solution. Following hybridization and washing, the signal can be detected by chemiluminescent detection. The purified DNA band can also be used as a probe for non-isotopic cDNA library screening to clone full-length cDNA of the gene. The purified band can be sequenced directly using the same biotinylated oligo( dT)-anchored primer and SeqLight@ system. After DNA sequence data are obtained, primers can be designed and used in a relative reverse transcription-PCR (RT-PCR) experiment to confirm the differential expression of the gene, or used in a Rapid Amplification of cDNA End (RACE) experiment to clone the fu11length cDNA of the gene.

Differential Display Polymerase Chain Reaction Using Chemiluminescent 13.3

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AND DISCUSSION

The described method is applied to identify genes differentially expressed between normal prostate and prostate cancer tissues. Figure 13.2 shows an example of the sequencing gel from the differential display experiments using chemiluminescent detection. In this particular experiment, biotinylated T 12VG

primer is used together with either OPB 1 or OPB2 primer (Operon Technologies). The bands

OPBl N

C

VI

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FIGURE 13.2 DD band patterns by chemiluminescent detection. Primers used: 5'-biotin-llllllllllllVG-3' and OPB 1: 5' -GTITCGCTCC-3' or OPB2: 5' -TGATCCCTGG-3'. N, RNA from normal prostate tissue; C, RNA from prostate cancer tissue; arrows indicate differentially expressed bands.

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DI

VI

Actin

FIGURE 13.3 Confirmation of differential expression by RT-PCR. The results of RT-PCR for DI and UI transcripts and f3-actincontrol are shown. N, normal prostate tissues; C, prostate cancer tissue. Upper panel shows the RT-PCR result of DI transcript, middle panel shows the RT-PCR result of UI transcript, and lower panel shows the f3-actincontrol.

indicated by the arrows represent differentially expressed bands. Whereas the DI band shows much higher intensity in normal prostate tissue, the V I band shows much higher intensity in prostate cancer tissue. Overall, the quality, sensitivity, and the total number of bands displayed are very similar to that obtained by radioisotope detection method (data not shown). We routinely obtained 50 to 200 bands per reaction for each primer set. Although most of the bands show identical intensity among the tissues examined, one to three differentially expressed bands are seen from each experiment by a primer set. Both the Dl and VI bands were recovered and purified as described. Half the purified DNA was used as a template for chemiluminescent sequencing. Primers were then designed for both genes based on sequence information, and RT-PCR11was performed on six each of normal and prostate cancer tissues to confirm the differential expression of the genes. j3-Actin RT-PCR was also performed as a control for normalization. As shown in Figure 13.3, D I was confirmed to be downregulated in prostate cancer, and V I was confirmed to be upregulated in prostate cancer. The confirmation rate of the differentially expressed cDNAs using our method is similar to that of DD using radioactive detection (data not shown). In summary, the DD-PCR using a chernilurninescentdetection method described here is a sensitive, reliable, cost-effective, and safe alternative to the radioisotopic differential display technique.3.4 Also, it does not require any costly special equipment or software. Since no radioisotopes are involved, all the safety and waste disposal issues related to radioactive material handling requirements are eliminated. In our hands it has the same sensitivity, reproducibility, and confirmation rate as the original radioisotope-based DD-PCR method.

ACKNOWLEDGMENTS We thank Dr. S. Mark O'Hara for helpful input during development of this method, and Ms. Guizhen Luo for providing technical assistance.

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REFERENCES 1. Sambrook, 1., Fritsch, E. F., and Maniatis, T., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. 2 Lee, S., Tomasetto, C., and Sager, R., Positive selection of candidate tumor suppressor gene by subtractive hybridization, Proc. Natl. Acad. Sci. U.S.A., 88,2825, 1991. 3. Liang, P. and Pardee, A. B., Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction, Science, 257, 967, 1992. 4. Liang, P., Averbouk, L., Keyomarsi, K., and Sager, R., Differential display and cloning of mRNAs from human breast cancer versus mammary epithelial cells, Cancer Res., 52, 6966, 1992. 5. Welsh, J., Chada, K., Dalal, S. S., Cheng, R., Ralph, D., and McClelland, M., Arbitrarily primed PCR fingerprinting of RNA, Nuc/. Acids Res., 20, 4965, 1992. 6. Reddy, P. M. S., An, G., Di, Y. P., Zhao, Y. H., and Wu, R., A palindromic primer-based mRNA differential display method to isolate vitamin A-responsive genes in airway epithelium: characterization of nucleolin gene expression. Am. J. Respir. Cell Mol. BioI., 15,398, 1996. 7. Lohmann, J., Schickle, H., and Bosch, T. C. G., REN display, a rapid and efficient method for nonradioactive differential display and mRNA isolation, BioTechniques, 18,200, 1995. 8. Smith, N. R., Aldersley, M., Li, A., High, A. S., Moynihan, T. P., Markham, A. F., and Robinson, P. A., Automated differential display using a fluorescent labeled universal primer, BioTechniques, 23,274, 1997. 9. An, G., Luo, G. Z., Veltri, R. w., and O'Hara, S. M., A sensitive, non-radioactive differential display method using chemiluminescent detection, BioTechniques, 20, 342, 1996. 10. Chomczynski, P. and Sacchi, N., Single-step of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction, Anal. Biochem., 162, 156, 1987. 11. An, G., Cazares, L., Luo, G., Miller, M. C., Wright, G. L., Jr., and Veltri, R. w., Differential expression of full length and a truncated Her-2/neu oncogene receptor in prostate cancer assessed using relative quantitative RT-PCR,Mol. Urol., 2,305, 1999.