Methods of high integrity RNA extraction from cell/agarose construct.

Takahiro Ogura,Akihiro Tsuchiya,Tom Minas,Shuichi Mizuno

doi:10.1186/s13104-015-1627-5

Takahiro Ogura, Akihiro Tsuchiya + Show 2 more

Open Access

https://doi.org/10.1186/s13104-015-1627-5

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Abstract

BackgroundAgarose 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.ResultsWith 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.ConclusionsThe 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.

Highlights

Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology
Concentration, purity, and integrity of the extracted RNA We evaluated the concentration, purity, and integrity of RNA extracted with various extraction methods from the Bovine articular chondrocytes (bACs)/agarose hydrogel construct
The bACs produced a metachromatic matrix around the cells, representing cartilage extracellular matrix (ECM)

Summary

Introduction

Agarose hydrogels are widely used for three-dimensional cell scaffolding in tissue engineering and cell biology. Little is known about RNA integrity when the RNA is extracted from cell/agarose construct. 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. Agarose hydrogels have been conveniently used for threedimensional (3-D) cell scaffolding. This is largely because the gels promote the maintenance of chondrogenic phenotypes, e.g., the synthesis of cartilaginous extracellular matrix (ECM), for neocartilage engineering and cell biology applications [1,2,3]. Gene expression profiles have begun to provide useful information for analyzing the activities of various genes To obtain those profiles, high quality RNA was required. RNA integrity using spectrophotometry and microfluidic serum, 100 units/ml penicillin and 100 μg/ml streptomycapillary electrophoresis.

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