Bactin primers (GTGGGGCGCCCCAGGCACCCA, CTCCTT AATGTCACGCACGATTTC) as a housekeeping gene. RNA of

Bactin primers (GTGGGGCGCCCCAGGCACCCA, CTCCTT AATGTCACGCACGATTTC) as a housekeeping gene. RNA of the same sample was used as negative control for detection of DNA contamination and DEPC treated water as non-template control.PCR Detection of CD44 Variable ExonsThe PCR reaction mixture contained12,5 ml AmpliTaq GoldH 360 Master Mix, 2.5?.5ml of the appropriate MedChemExpress SR-3029 primer pair designed with Array Designer (Premier Biosoft International) (Figure S1). 2ml of the cDNA and 5.5 ml DEPC treated water for the final volume of 25 ml. The cycling conditions were: 97uC for 10 min once, then 95uC for 1 min, 55uC for 1 min, 72uC for 2 min for 35 cycles, 72uC for 10 min. The primer pairs were the following: S5′- variable exons3′, variable exons5′-S3′, PCR products were separated using ExperionTM Automated DNA 1K Kit1ml (Bio-RadH) Electrophoresis System.Materials and Methods Cell Lines and Culture ConditionsThe A2058 melanoma cell line was provided by LA Liotta (NCI, Bethesda, MD). HT168 and HT168M1 lines are derivatives 18325633 of A2058 [31]. HT199 [31] was developed in the 1st Department of Pathology and Experimental Cancer Research (Semmelweis University, Budapest, Hungary). WM983B [32] and WM35 [32] were gifts from M. Herlyn (Wistar Institute, Philadelphia, PA). The colorectal carcinoma cell lines were HT25 [33] (from M. Hendricks, Iowa), HT29 (ECACC 91072201), HCT116 (ICLC HTL95025) and HCR31 [34]. We also used MDA-MB-231 human breast adenocarcinoma (ECACC 92020424), PE/CA PJ15 (ECACC 96121230) and PE/CA PJ41 (ECACC 98020207) human oral squamous cell TA 02 carcinomas, K562 human chronic myelogenous leukaemia (ECACC 89121407) and A431 human vulva squamous carcinoma (ECACC 85090402) cell lines. The melanocyte (C-12403), keratinocyte (C-12003) and fibroblast (C-12360) cells were derived from Promo Cell. The melanoma and colorectal cell lines were maintained in RPMI 1640 medium supplemented with 10 fetal bovine serum (Sigma, St. Louis, MD), 2 mMPCR Detection and Sequencing of CD44 FingerprintThe PCR conditions were as described above. The primer pairs were the following: S5′- S3′, S5′- v33′, v35′- S3′, S5′- v63′, v35’v63′ using the exon specific primers (Figures S1, S2 and S3). PCR products were separated on a 3 agarose gel and detected with Gel Doc 2000 (Bio-RadH) after ethidium bromide staining. PCR products were re-isolated from the agarose gel (High Pure PCR Product Purification Kit, Roche, Mannheim) in the case of all bands. The DNA sequences were determined by BigDyeH Terminator v1.1 Cycle Sequencing Kit (Applied BiosystemsTM ?by Life TechnologiesTM).Cloning the PCR Products from the FingerprintThe PCR products of the fingerprint were re-isolated from the 2 agarose gel (as above) inserted into vectors in the pGEMH-T Vector Systems (Promega H) according to the manufacturer’s instructions. The plasmid DNA was re-isolated using GeneJET (TM) Plasmid Miniprep Kit (Thermo Scientific) according to the manufacturer’s instructions and the products were sequenced with BigDyeH Terminator v1.1 Cycle Sequencing Kit (Applied BiosystemsTM ?by Life TechnologiesTM).CD44 Alternative Splicing Pattern of MelanomaNext-generation SequencingDuring a library preparation the ligated amplicon generation was used. The PCR products from HT199, A2058 and WM983B melanoma fingerprints were amplified, purified and their ends were polished and ligated with Roche 454 multiplex identifier (MID) adaptors, to generate universal primer binding sites for emulsion PCR in every sample reactions. The a.Bactin primers (GTGGGGCGCCCCAGGCACCCA, CTCCTT AATGTCACGCACGATTTC) as a housekeeping gene. RNA of the same sample was used as negative control for detection of DNA contamination and DEPC treated water as non-template control.PCR Detection of CD44 Variable ExonsThe PCR reaction mixture contained12,5 ml AmpliTaq GoldH 360 Master Mix, 2.5?.5ml of the appropriate primer pair designed with Array Designer (Premier Biosoft International) (Figure S1). 2ml of the cDNA and 5.5 ml DEPC treated water for the final volume of 25 ml. The cycling conditions were: 97uC for 10 min once, then 95uC for 1 min, 55uC for 1 min, 72uC for 2 min for 35 cycles, 72uC for 10 min. The primer pairs were the following: S5′- variable exons3′, variable exons5′-S3′, PCR products were separated using ExperionTM Automated DNA 1K Kit1ml (Bio-RadH) Electrophoresis System.Materials and Methods Cell Lines and Culture ConditionsThe A2058 melanoma cell line was provided by LA Liotta (NCI, Bethesda, MD). HT168 and HT168M1 lines are derivatives 18325633 of A2058 [31]. HT199 [31] was developed in the 1st Department of Pathology and Experimental Cancer Research (Semmelweis University, Budapest, Hungary). WM983B [32] and WM35 [32] were gifts from M. Herlyn (Wistar Institute, Philadelphia, PA). The colorectal carcinoma cell lines were HT25 [33] (from M. Hendricks, Iowa), HT29 (ECACC 91072201), HCT116 (ICLC HTL95025) and HCR31 [34]. We also used MDA-MB-231 human breast adenocarcinoma (ECACC 92020424), PE/CA PJ15 (ECACC 96121230) and PE/CA PJ41 (ECACC 98020207) human oral squamous cell carcinomas, K562 human chronic myelogenous leukaemia (ECACC 89121407) and A431 human vulva squamous carcinoma (ECACC 85090402) cell lines. The melanocyte (C-12403), keratinocyte (C-12003) and fibroblast (C-12360) cells were derived from Promo Cell. The melanoma and colorectal cell lines were maintained in RPMI 1640 medium supplemented with 10 fetal bovine serum (Sigma, St. Louis, MD), 2 mMPCR Detection and Sequencing of CD44 FingerprintThe PCR conditions were as described above. The primer pairs were the following: S5′- S3′, S5′- v33′, v35′- S3′, S5′- v63′, v35’v63′ using the exon specific primers (Figures S1, S2 and S3). PCR products were separated on a 3 agarose gel and detected with Gel Doc 2000 (Bio-RadH) after ethidium bromide staining. PCR products were re-isolated from the agarose gel (High Pure PCR Product Purification Kit, Roche, Mannheim) in the case of all bands. The DNA sequences were determined by BigDyeH Terminator v1.1 Cycle Sequencing Kit (Applied BiosystemsTM ?by Life TechnologiesTM).Cloning the PCR Products from the FingerprintThe PCR products of the fingerprint were re-isolated from the 2 agarose gel (as above) inserted into vectors in the pGEMH-T Vector Systems (Promega H) according to the manufacturer’s instructions. The plasmid DNA was re-isolated using GeneJET (TM) Plasmid Miniprep Kit (Thermo Scientific) according to the manufacturer’s instructions and the products were sequenced with BigDyeH Terminator v1.1 Cycle Sequencing Kit (Applied BiosystemsTM ?by Life TechnologiesTM).CD44 Alternative Splicing Pattern of MelanomaNext-generation SequencingDuring a library preparation the ligated amplicon generation was used. The PCR products from HT199, A2058 and WM983B melanoma fingerprints were amplified, purified and their ends were polished and ligated with Roche 454 multiplex identifier (MID) adaptors, to generate universal primer binding sites for emulsion PCR in every sample reactions. The a.