Differential display PCR (DD-PCR) is an mRNA fingerprinting technique to identify differentially expressed genes by comparative display of arbitrarily amplified cDNA subsets. This attractively simple screening method was, however, followed by a labour intensive multistep identification procedure for DD-PCR products. In this report we demonstrate for the mouse mast cell protease 2 (MMCP-2) and the cytotoxic T-lymphocyte associated gene transcript CTLA-1 a streamlined approach by (i) direct cycle sequencing with the upstream differential display (DD) primer, followed by (ii) the PCR based generation of an antisense northern probe with the downstream anchor prime

Buess, Martin

Hirsch, Hans H.

Moroni, Christoph

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 1997 Oxford University Press 2233–2235Nucleic Acids Research, 1997, Vol. 25, No. 11Direct identification of differentially expressed genesby cycle sequencing and cycle labelling using thedifferential display PCR primersMartin Buess, Christoph Moroni and Hans H. Hirsch*Institute for Medical Microbiology, University of Basel, Petersplatz 10, 4003 Basel, SwitzerlandReceived February 21, 1997; Revised and Accepted April 9, 1997ABSTRACTDifferential display PCR (DD-PCR) is an mRNA finger-printing technique to identify differentially expressedgenes by comparative display of arbitrarily amplifiedcDNA subsets. This attractively simple screeningmethod was, however, followed by a labour intensivemultistep identification procedure for DD-PCR products.In this report we demonstrate for the mouse mast cellprotease 2 (MMCP-2) and the cytotoxic T-lymphocyteassociated gene transcript CTLA-1 a streamlinedapproach by (i) direct cycle sequencing with theupstream differential display (DD) primer, followed by(ii) the PCR based generation of an antisense northernprobe with the downstream anchor primer.We have applied differential display PCR (DD-PCR; 1) to amurine tumor model in which transduction of the v-H-rasoncogene into IL-3 dependent non tumorigenic PB-3c mast cellsgave rise to IL-3 autocrine tumors with an increased IL-3 mRNAstability. IL-3 mRNA expression from this autocrine tumor couldbe suppressed by somatic cell fusion with precursor cells implyinga recessive alteration (2–4).Culturing of the precursor cell PB-3c clone 20, the tumor lineV2D1 and the hybrid 20/V2D1 has been described previously (3).Total cytoplasmic RNA was isolated from each cell line accordingto Gough (5) followed by DNAse I digestion (6). Integrity of theRNA was checked by agarose formaldehyde gel electrophoresis.Comparison of precursor PB-3c clone 20 with the tumor V2D1cells was performed by the DD-PCR technique essentially asdescribed by Bauer et al. (7). For reverse transcription of an mRNAsubset, 0.2 µg of RNA was mixed first with 50 pmol anchor primer,dT12VN, (V representing A, G or C and N being A, C, G or T) ina volume of 7 µl, kept for 10 min at 65C and immediately put onice. Following addition of 300 U MMLV- reverse transcriptase(Gibco Life Technologies) it was incubated for 1 h at 37C inreverse transcription buffer (40 mM KCl, 50 mM Tris–HCl pH 8.3,6 mM MgCl2) containing 20 µM dNTP, 10 mM DTT and 40 URNAsin (Boehringer) in a total volume of 20 µl. After 5 minat 95C the cDNA was stored at –20C until use in DD-PCR.DD-PCR was performed with 2 µl of 20 µl cDNA in a Perkin-Elmer Cetus thermal cycler 480 using PCR buffer (10 mM Tris–HClpH 8.3, 50 mM KCl), 2.5 U AmpliTaq DNA Polymerase (PerkinElmer), 1.25 mM MgCl2, 4 µM dNTP, 1 µCi [α-32P]dCTP, 2.5 µMof the respective downstream anchor primer dT12VN and 0.5 µMof one upstream DD-primer from the set of 26 decamericoligonucleotide primers according to Bauer et al. (7) using thefollowing conditions: 5 min incubation at 95C followed by 40cycles of 94C for 30 s, 40C for 2 min and 72C for 30 s. PCRproduct (3 µl) was added to 2 µl sample buffer and incubated at95C for 5 min. The PCR products were resolved on a 6%polyacrylamide–7 M urea gel in 1× TBE buffer (89 mM Tris,89 mM boric acid, 0.025 mM EDTA). The gel was dried andanalysed by a PhosphorImager  (Molecular Dynamics) (Fig. 1Aand B).The differentially displayed bands were cut out from a wetpolyacrylamide gel after localisation on a X-ray film. The DNAwas eluted with the QIAEX II Gel Extraction Kit (QIAGEN)according to the protocol of the supplier. With 6 µl of the 20 µleluate the DD-PCR was repeated without labelled dCTP but with100 µM dNTP. The reamplification product was resolved on a 2%TBE agarose gel, stained with ethidium bromide (Fig. 1C),recovered with the QIAEX II Gel Extraction Kit and resuspendedin 20 µl ddH2O. This amplicon was used for (i) direct sequencingand (ii) generation of a labelled probe.Direct sequence determination with the upstream DD-primerwas done on the ABI 310 genetic analyzer using the ABIPRISM Dye Terminator Cycle Sequencing Ready ReactionKit (Perkin Elmer). Gel purified reamplification product (6 µl)was mixed with 32 pmol upstream DD-primer and 8 µl terminatorready reaction mix in a total volume of 20 µl. We modified thecycling conditions for the sequencing with short decamericDD-primers as follows: 25 cycles of 96C for 30 s, 40C for 15 sand 60C for 4 min (480 Perkin Elmer Cetus Thermal Cycler).The samples were prepared for the sequence analysis as describedby the supplier using the ethanol precipitation protocol to removeunincorporated dye terminators (Fig. 1D and E). Sequencedetermination of 100–150 nt allowed the search in the gene bankand direct cycle labelling for northern analysis.For northern analysis, an antisense probe was generated bycycle labelling with the downstream anchor primer. Gel purifiedreamplification product as template (5 µl) was incubated withPCR buffer (10 mM Tris–HCl pH 8.3, 50 mM KCl), 25 pmol ofthe respective T12VN, 0.04 mM dNTP, 50 µCi [α-32P]dCTP and2.5 U AmpliTaq DNA Polymerase  (Perkin Elmer) in a total*To whom correspondence should be addressed. Tel: +41 61 267 3292; Fax: +41 61 267 3298; Email: hirsch@ubaclu.unibas.ch Nucleic Acids Research, 1997, Vol. 25, No. 112234Figure 1.  Illustration of the direct cycle sequencing and cycle labelling strategy for DD-PCR products with two differentially expressed genes as described in the text.volume of 100 µl (5 min at 95C, 40 cycles of 1 min at 96C, 2 minat 35C, 1 min at 72C). The labelled product was extracted withthe QIAquick PCR Purification Kit (QIAGEN) and the integrityand size of the probe was visualised on a 6% polyacrylamide–7 Murea gel (Fig. 1F). Hybridisation was done with 6 × 106 c.p.m. ina volume of 5 ml. Northern blots were prepared as describedpreviously with 20 µg RNA per lane (8). The filters wereprehybridised in 5 ml prewarmed hybridisation solution (50%formamide, 5× SSC, 5× Dehnhardt’s solution, 5 mM EDTA, 0.5mg/ml yeast tRNA, 0.1 mg/ml heparin, 0.2% SDS) for 2 h at 45Cin a rotating hybridisation oven. After adding the heat denaturedprobe, the filter was hybridised overnight. Washing was donetwice with prewarmed 2× SSC, 0.1% SDS at 55C for 30 min.The filter was analysed by a PhosphorImager (Fig. 1G and H).To illustrate this approach we show the identification of oneupregulated (Fig. 1A) and one downregulated band (Fig. 1B)which were found during DD-PCR comparison of the tumor lineV2D1 and the precursor line PB-3c clone 20 with the followingprimer pairs: anchor primer T12CG (T2) versus DD-primer D13(GTTTTCGCAG) and anchor primer T12CC (T4) versus DD-primer D5 (GGAACCAATC). As shown in Figure 1D and E, thereamplified DD-PCR products could be directly sequenced usingDD-primers D13 or D5, respectively. The sequences could beread over a stretch of more than 100 nt in sense direction at the2235Nucleic Acids Research, 1994, Vol. 22, No. 1Nucleic Acids Research, 1997, Vol. 25, No. 11 2235end of which the complementary sequence to the respectivedownstream anchor primer could be identified (Fig. 1E).Comparison of both sequences with entries in GenBank/EMBL/PDB databases identified a 100% match with the mouse mast cellprotease 2 (MMCP-2) (9) and the mouse cytotoxic T lymphocyteassociated gene transcript CTLA-1 (10), respectively. Differentialexpression of both genes was confirmed by northern analysisusing the antisense probe generated from the sequenced fragments.MMCP-2 is expressed in the tumor cell V2D1, but is notexpressed in the precursor cell. The expression of MMCP-2 in thehybrid 20/V2D1 is at least 10 times downregulated compared tothe tumor cell (Fig. 1G). Ethidium bromide stained gels served asloading controls. Initially identified in Ki-ras transformed mastcells, MMCP-2 is a marker for intermediate differentiation stagesbetween the bone marrow derived and the fully mature mast cells(9). In contrast CTLA-1 is expressed in the precursor cell PB-3cclone 20 but is missing in the tumor V2D1 and in the hybrid cell(Fig. 1H). This serine protease belongs to the granzyme grouppredominantly expressed in cytotoxic T-cells but it has also beenfound in mast cells, yet is lacking in the mastocytoma P815 (10).Direct cycle sequencing with decameric DD-primers and directgeneration of an antisense probe by cycle labelling is a streamlinedapproach to identify a differentially expressed gene representedby a DD-PCR fragment. It avoids the conventionally used labourintensive subcloning procedure, which consisted of ligation intoa plasmid vector, transformation of Escherichia coli cells andidentification of a clone corresponding to the differentiallyexpressed gene before the sequence could be determined. Thecosts for the modification of the DD-PCR product by primerextension in order to use classical sequencing primers for directsequencing can also be saved (11). In the optimal case thesequence and the confirmation of differential expression can beobtained within 3 days. If the reamplified DD-PCR productconsists of more than one template or was primed by the sameprimer on both sides subcloning cannot be avoided, which was thecase in ∼20% of the 30 differentially displayed bands detectedduring our systematic DD-PCR screening. Furthermore, the directidentification of DD-PCR products generated with different primercombinations but arising from the same differentially expressedgene avoids unnecessary further analysis. If northern hybridisationdoes not yield a signal due to low abundance of the transcript, thesequence may provide the information for other more sensitivemethods to confirm differential expression.ACKNOWLEDGEMENTSM. Buess is a MD-PhD fellow of the Roche Research Foundation.This work was supported in part by the grant 31-40816.94 of theSchweizerische National Fonds zur Förderung der Wissenschaft-lichen Forschung.REFERENCES1 Liang, P. and Pardee,A.P. (1992) Science, 257, 967–971.2 Nair,A.P.K., Diamantis,I.D., Conscience,J.-F., Kindler V., Hofer,P. andMoroni,C. (1989) Mol. Cell. Biol., 9, 1183–1190.3 Hirsch,H.H., Nair,A.P.K. and Moroni,C. (1993) J. Exp. Med., 178,403–411.4 Hirsch,H.H., Nair,A.P.K., Backenstoss,V. and Moroni,C. (1995) J. Biol.Chem., 270, 20629–20635.5 Gough,N.M. (1987) Anal. Biochem., 173, 93–95.6 Liang,P., Averboukh,L. and Pardee,A.B. (1993) Nucleic Acids Res., 21,3269–3275.7 Bauer,D., Müller,H., Reich,J., Riedel,H., Ahrenkiel,V., Wartoe,P. andStrauss,M. (1993) Nucleic Acids Res., 21, 4272–4280.8 Thomas.P. (1980) Proc. Natl. Acad. Sci. USA, 77, 5201–5205.9 Serafin,W.E., Reynolds,D.S., Rogelj,S., Lane,W.S., Conder,G.A.,Johnson,S.S., Austen,F.A. and Stevens,R.L. (1990) J. Biol. Chem., 265,423–429.10 Brunet,J.-F., Denizot,F., Suzan,M., Haas,W., Mencia-Huerta,J.-M.,Berke,G., Luciani,M.-F. and Golstein,P. (1987) J. Immunol., 138,4102–4105.11 Wang,X. and Feuerstein,G.Z. (1995) BioTechniques, 18, 448–453.

Direct identification of differentially expressed genes by cycle sequencing and cycle labelling using the differential display PCR primers

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