title: Methods of high integrity RNA extraction from cell/agarose construct.
abstract: BACKGROUND
  Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology. Recently, molecular profiles have been obtained with extraction of a minimal volume of RNA using fluorescent-tagged quantitative polymerase chain reaction (qPCR), which requires high integrity RNA. However, the agarose interferes considerably with the quantity and quality of the extracted RNA. Moreover, little is known about RNA integrity when the RNA is extracted from cell/agarose construct. Thus, in order to obtain RNA of sufficient integrity, we examined various extraction methods and addressed reproducible methodologies for RNA extraction from cell/agarose constructs using spectrophotometry and microfluidic capillary electrophoresis.
  
  
  RESULTS
  With various extraction methods using a mono-phasic solution of phenol and guanidine isothiocyanate, we evaluated quantity and quality of total RNA from cell/agarose construct. Extraction with solution of phenol and guanidine isothiocyanate followed by a silica based membrane filter column gave sufficient RNA integrity number, which allowed us to proceed to fluorescent-tagged qPCR for evaluating various cellular activities.
  
  
  CONCLUSIONS
  The RNA extraction methods using phenol and guanidine isothiocyanate solution and a silica membrane column can be useful for obtaining high integrity RNA from cell/agarose constructs rich in polysaccharide and extracellular matrix. Our study contributes to further investigation using agarose hydrogels and other materials rich in polysaccharide in the field of cellular and tissue engineering.
authors.affiliation: Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA, 02115, USA. togura@partners.org.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ogura
authors.affiliation: Funabashi Orthopaedic Hospital Sports Medicine Center, 1-833, Hasamacho, Funabashi, Chiba, 274-0822, Japan. a-tsuchiya@fff.or.jp.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Tsuchiya
authors.affiliation: Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA, 02115, USA. tminas@partners.org.
authors.forename: Tom
authors.initials: T
authors.lastname: Minas
authors.affiliation: Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA, 02115, USA. smizuno@rics.bwh.harvard.edu.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Mizuno
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D006146:Guanidines; D017879:Isothiocyanates; D046210:Microfluidic Analytical Techniques; D008967:Molecular Biology / Q000379:methods; D019800:Phenol; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012685:Sepharose; D013053:Spectrophotometry
chemical_list: D006146:Guanidines; D017879:Isothiocyanates; C054435:guanidine isothiocyanate; D019800:Phenol; D012313:RNA; D012685:Sepharose