Methods of high integrity RNA extraction from cell/agarose construct

BMC Research Notes

Shuichi Mizuno²  Tom Minas²  Akihiro Tsuchiya¹  Takahiro Ogura² 

[1] Funabashi Orthopaedic Hospital Sports Medicine Center, 1-833, Hasamacho, Funabashi 274-0822, Chiba, Japan;Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis St., Boston 02115, MA, USA

关键词: Tissue engineering;    Polysaccharide;    Chondrocyte;    RNA integrity;    RNA;    Agarose gel;   

Others  :  1232824
DOI  :  10.1186/s13104-015-1627-5

 received in 2015-06-09, accepted in 2015-10-26,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Ogura et al.

【 图 表 】

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