title: Preparation of native and amplified tumour RNA for dendritic cell transfection and generation of in vitro anti-tumour CTL responses.
abstract: Recent research indicates that dendritic cells transfected with RNA-encoded tumour-associated antigens (TAA) can generate potent anti-tumour immune responses in vitro and in vivo. RNA is an important source of TAA, but its relatively unstable nature, in addition to often limited availability of tumour tissue, may represent a considerable obstacle for its use. Our first goal was to establish an efficient protocol for the preparation of high quality total RNA from tumour samples. This should then be used as such or be pre-amplified for DC transfection. Therefore native total RNA was prepared from stabilised tissue samples obtained from liver metastases of colon cancer using either solution- or silicagel-based protocols for RNA isolation. The first isolation protocol yielded higher amounts of total RNA, but with lower purity as compared to the second one. No degradation of RNA was observed regardless of the protocol used. Subsequently, we focused on the amplification of mRNA. The fidelity of the amplified mRNA was confirmed by RT-PCR for glyceraldehyde-3-phosphate-dehydrogenase (GADPH) and carcinoembryonic antigen (CEA) coding sequences. We found no differences in the induction of CEA-specific CTL responses between native and amplified RNA-transfected DCs. Additionally, we tested the induction of CTL responses and found that DCs transfected with amplified mRNA originating from either tumour tissue or a cell line were able to induce strong anti-tumour CTL responses in vitro. They were comparable to those induced by native total RNA-transfected DCs. Our results therefore indicate that the amplified mRNA is equivalent to the native one in the induction of anti-tumour CTL responses and can be used for generation of RNA-transfected DCs.
authors.affiliation: Tissue Typing Center, Blood Transfusion Centre of Slovenia, Ljubljana, Slajmerjeva 6, SI-1000 Ljubljana, Slovenia. martina.bergant@gmail.com
authors.forename: Martina
authors.initials: M
authors.lastname: Bergant
authors.forename: Luka
authors.initials: L
authors.lastname: Meden
authors.forename: Urska
authors.initials: U
authors.lastname: Repnik
authors.forename: Valentin
authors.initials: V
authors.lastname: Sojar
authors.forename: Dragoje
authors.initials: D
authors.lastname: Stanisavljević
authors.forename: Matjaz
authors.initials: M
authors.lastname: Jeras
mesh_terms: D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D003713:Dendritic Cells / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009369:Neoplasms / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D021141:Nucleic Acid Amplification Techniques; D012333:RNA, Messenger / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D012334:RNA, Neoplasm