Preparation of native and amplified tumour RNA for dendritic cell transfection and generation of in vitro anti-tumour CTL responses

Immunobiology
Martina BergantMatjaz Jeras

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 ampl...Continue Reading

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Citations

May 7, 2009·The Journal of Membrane Biology·Mateja GabrijelRobert Zorec
May 16, 2007·Cancer Immunology, Immunotherapy : CII·Sandra TuyaertsAude Bonehill
Mar 5, 2009·Cancer Immunology, Immunotherapy : CII·Catherine L RileyRobert C Rees
Aug 24, 2012·PLoS Pathogens·Sophia MaschalidiBénédicte Manoury
Sep 17, 2013·PloS One·Ryuichi MurakamiTatsuro Irimura
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Mar 4, 2014·Clinica Chimica Acta; International Journal of Clinical Chemistry·Outi Itkonen, Ulf-Håkan Stenman

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