Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis.
For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A260 values (representing up to 40.8 μg RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A260/A280 and A260/A230 ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low Ct values of ∼20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen , Giessen, Germany .
Natascha
N
Köster
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen , Giessen, Germany .
Alexandra
A
Schmiermund
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen , Giessen, Germany .
Stefan
S
Grubelnig
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen , Giessen, Germany .
Jasmin
J
Leber
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen , Giessen, Germany .
Franziska
F
Ehlicke
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen , Giessen, Germany .|2 Department of Chemical Engineering, Kansas State University , Manhattan, Kansas.|3 Faculty of Chemistry and Biology, University of Giessen , Giessen, Germany .|4 Project Group Bioresources, Fraunhofer Institute for Molecular Biology an Applied Ecology IME , Giessen, Germany .
Peter
P
Czermak
1 Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen , Giessen, Germany .
Denise
D
Salzig
D002478:Cells, Cultured; D002593:Cetrimonium Compounds / Q000737:chemistry; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D023822:Tissue Engineering / Q000379:methods*
D002593:Cetrimonium Compounds; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D012313:RNA