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Construction of a Representative Cdna Library from Prostatic Intraepithelial Neoplasia

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Construction of a Representative Cdna Library from Prostatic Intraepithelial Neoplasia ICANCERRESEARCH56. 5380-5383. December 1. 19961 Advances in Brief Construction of a Representative cDNA Library from Prostatic Intraepithelial Neoplasia David B. Krizman,' Rodrigo F. Chuaqw, Paul S. Meltzer, Jeffrey M. Trent, Paul H. Duray, W. Marston Linehan, Lance A. Liotta, and Michael R. Emmert-Buck Laboratory of Cancer Genetics ID. B. K.. P. S. M., J. M. TI, National Centerfor Human Genome Research, Laboratory of Pathology (R. F. C., P. H. D., L A. L, M. R. E-B.J, and Surgical Branch fW. M. LI. National Cancer Institute, Bethesda, Maryland 20892 Abstract cDNA libraries corresponding to cells derived exclusively from a microscopic prostatic lesion (PIN). Our results indicate that construc We report the construction of a plasmid-based eDNA library made tion of cDNA libraries arising from cells of prostate-specific epithelial from microdissected cells derived from prostatic mtraepithelial neoplasia. origin can be achieved and should be of value in elucidating genes Total RNA was extracted and converted to blunt-ended, double-stranded eDNA by oligo(dT)-mediated reverse transcription followed by linker important in this disease. addition. A linker-specific primer with UDG.compatible ends was used to Materials and Methods amplify the eDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the Clinical Information and Microdissection. Tissue sample was obtained clones derived from either known genes, anonymous expressed sequence from a radical prostatectomy performed at the National Cancer Institute tags, or novel transcripts with very little redundancy of screened clones. (Bethesda, MD). Samples were snap frozen immediately after surgery and These results demonstrate the feasibility of constructing complex repre stored at —70°Cuntiluse. National Cancer Institute patient 1542 is a 47-year sentative eDNA libraries from specific microdissected cell populations old black male who presented with localized (clinical stage T2@)prostate that represent microscopic precursor stages of cancer progression. This cancer. The patient was clinically free of disease until 1 year postoperatively method should facilitate identification of transcripts specifically expressed when he developed a rapidly rising PSA and clinical evidence of recurrent in cells of a distinct histological origin and tumorlgemc stage. disease. Histopathological examination of the prostatectomy specimen showed Introduction a poorly differentiated adenocarcinoma (Gleason sum 8). Several small (<1 mm) discrete foci of PIN were present. cDNA libraries have proven essential to positional gene cloning Unstained l2-@tmfrozen tissue sections were dissected under microscopic efforts, differential gene expression studies, and EST2 sequencing! visualization as described previously (8, 9). A pure population of PIN cells was mapping projects. Traditionally, cDNA libraries are constructed from dissected. An adjacent H&E-stained section was used as a guide to ensure 1 to S ,.tg of purified mRNA obtained from either large heterogeneous accuracy of dissection. The PIN focus used for microdissection was selected based on defined histopathological criteria (10, 11). Approximately 2000— tissue samples or tissue culture cell lines (1—3).The technology of 5000 PIN cells were microdissected. cDNA library construction is relatively straightforward, given such RNA Extraction and Determination of RNA Integnty. A modified ver large amounts of starting material. However, because large tissue sion of the Stratageme RNA Microisolation (La Jolla, CA) procedure was used samples invariably contain substantial cellular heterogeneity, they are to isolate total cellular RNA. After resuspension of the RNA pellet, a DNase not a suitable source of starting material for generation of a represen step and reextractiomwas performed using the MessageClean protocol (Gene tational library from a distinct cell type. Also, because of limited Hunter, Nashville, TN) according to the manufacturer's recommendations.The tissue availability, precursor lesions of cancer (such as epithelial final RNA sample was resuspended in diethylpyrocarbomate-treated water dysplasia and in situ carcinoma) cannot be specifically studied from containing 0.5 units/id RNase inhibitor. bulk tissue specimens. Finally, it is not known whether gene expres The integrity of the RNA prep was determined with RT-PCR analysis of sion profiles of in vitro cell lines necessarily reflect the expression @3-actinmRNA.RT was perfonned utilizing the RNA map protocol according to the manufacturer's recommendations (GemeHunter),except 2.5 @zMrandom profiles of their tissues of origin, making this source of RNA also hexamers were used as primer. The final mixture was incubated as follows; problematic. Thus, it would be of considerable interest to generate 65°Cfor5 mm, 25°Cfor10mm, 1 j.tlMoloney murine leukemia virus reverse cDNA libraries directly from specific cell populations of various transcriptase added, 25°Cfor10 mm, 37°Cfor40 mm, and 94°Cfor5 mm. tissues to serve as reagents for studying in vivo gene expression. Each RT reaction generated 20 @.dofcDNA.Amplification ofactim mRNA was Although technologies designed to generate complex and represent performed using 1 @lofthe cDNA sample with actin-specific primers (Clon ative cDNA libraries from small amounts of total cellular RNA of a tech, Palo Alto, CA). The reaction was run twice to ensure reproducibility of given cell type have previously been developed (4—7),they would results and a negative reverse transcription reaction was run in parallel. benefit from pathology techniques enriching for the desired type of eDNA Synthesis and Library Construction. Double-strandedcDNAwas diseased cell. synthesized from 0.04 @gof total cellular RNA using the Superscript Choice We now report the feasibility of combining microdissection of System based on the RNase H-mediated second-strand replacement method (2), according to the manufacturer's recommendations, with some modifica specific cell populations with cDNA library construction to generate tions (Life Technologies, Inc., Gaithersburg, MD). RT was carried out in a l0-pJ volume reaction. A concentration of 50 ng/pi oligo(dT) was used for Received 8/9/96; accepted 10/I7/96. priming first-strand cDNA, and this reaction was carried out at 45°Cfor15ruin The costs of publication of this article were defrayed in part by the payment of page instead of at the recommended 37°Cfor 30 mm. The second-strand replace charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ment and EcoRI linker addition reactions were followed according to the I To whom requests for reprints should be addressed, at NCHGRINIH Building 49, manufacturer's suggestions. After linker addition, the double-stranded cDNA Room 4A15. 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 402-2015; Fax: was electrophoresed in 1.2% low-melt agarose and products from 0.3 to 2 kb (301) 402-3241. were excised with a scalpel blade. The agarose was melted at 65°Cfor15 rain, 2 The abbreviations used are: EST, expressed sequence tag; PIN, prostatic intraepithe hal meoplasia;RT, reverse transcription; PSA, prostate-specific antigen; dbEST, database digested overnight at 37°Cwith(3-agaroseenzyme according to the manufac of expressed sequence tags. turer's recommendations (New England BioLabs, Beverly, MA), phenol/ 5380 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. CONSTRUCTION OF A cDNA LIBRARY FROM PIN chloroform extracted, and ethanol precipitated. The cDNA pellet was resus Table 1 CumulativeclonesNo.sequence analysis of 154PiN-specific cDNA pended in 20 @lTEand stored at —20°C. clonesIdentification(n of A total of 5 pAof the cDNA was amplifiedby five cycles of PCR under totalRedundanciesNoinsert1610rRNA138.5Anonymous154)% of standard reaction conditions using 2.5 units of PerkimElmer-Cetus Taq po lymerase and 1.0 @.LMconcentrationof the linker-specific primer LINK (CUACUACUACUAAATrCGCGGCCGCGTCGAC) that also functions to eachKnown ESTs54352 ESTs 2X direct UDG cloning. The cycling conditions consisted of an initial 3-mm genes5334.5PSA 3X, ribosomal L29 2X, XUnique1812 cytochrome c oxidase 2 denaturing step at 94°Cfollowedby five cycles of94°Cfor15s, 55°Cfor15s, and 72°Cfor2 mm, with a final extension at 72°Cfor5 ruin.The PCR product Individual clone sequences were compared to sequence data bases using the BLAST was size selected by passing through a CHROMA SPIN-200 column to remove program. Identification groups are shown with total number of clones originating from the unincorporated primers, primer/primer artifacts, nucleotides, Taq, and buffers various groups calculated as well as the percentage of the total. The amount of redundancy according to the manufacturer's recommendations (Clontech). The flow is also shown for each group. Novel sequences showed no significant similarities to existing data base sequences; however, most demonstrated weak similarities to ESTs from through was ethanol precipitated, resuspended in 20 @.dofTE, and 6 @zl dbEST. nondirectionally cloned into the UDG cloning vector pAMPIO according to the manufacturer's recommendations (Life Technologies, Inc.). Recombinant clones were randomly picked for sequencing. Sequencing and Data Analysis. Clones were propagated and sequence Individual library subclones were picked at random for one pass ready DNA
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