Development of an RNA isolation procedure for the characterisation of human endothelial cell interactions with polyurethane cardiovascular bypass grafts.
To date no reliable method has been developed for the isolation of RNA from cells seeded onto cylindrical vascular grafts. This study was performed in order to develop a reliable methodology for isolating RNA from cylindrical conduits made from poly(carbonate-urea)urethane (PU). Human umbilical vein EC were seeded onto PU vascular grafts and an Alamar blue assay performed to assess cell viability. Cells were prepared for RNA extraction by trypsinisation, cell scraping and direct application of cell lysis buffer. In all cases RNA was extracted using a "Qiagen RNeasy" kit. Alamar blue showed viable cells were present on all of the seeded PU vascular grafts. Levels of RNA extracted from the cells removed from the graft by the trypsinisation yielded 0.130 microg/microl, by scraping 0.078 microg/microl and by direct lysing 0.093 microg/microl of RNA, respectively. RTPCR was conducted successfully for GAPDH and TGF-beta1. Trypsinisation prior to RNA extraction provided the highest RNA yield and attained near complete cell removal ensuring that gene expression obtained was representative.
Vara
Dina S
DS
Biomaterial & Tissue Engineering Centre, University Department of Surgery, University College London, Rolland Hill Street, Hampstead, London NW3 2PF, UK.
Punshon
Geoffrey
G
Sales
Kevin M
KM
Salacinski
Henryk J
HJ
Dijk
Sas
S
Brown
Robert A
RA
Hamilton
George
G
Seifalian
Alexander M
AM
D001672:Biocompatible Materials / Q000737:chemistry; D001705:Bioprosthesis; D001807:Blood Vessel Prosthesis; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008422:Materials Testing / Q000379:methods; D010078:Oxazines; D011108:Polymers / Q000737:chemistry*; D011140:Polyurethanes / Q000737:chemistry*; D012313:RNA / Q000302:isolation & purification*; D013048:Specimen Handling / Q000379:methods; D023822:Tissue Engineering / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D014966:Xanthenes
D001672:Biocompatible Materials; D010078:Oxazines; D011108:Polymers; D011140:Polyurethanes; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014966:Xanthenes; C110029:poly(carbonate urea) urethane; C005843:resazurin; D012313:RNA