A Simplified Method for RNA Isolation from Biofabricating Hydroxyapatite Scaffolds and Identification of Appropriate Reference Genes

Abstract

Abstract Purpose To validate a simplified RNA isolation method from biofabricating hydroxyapatite (HAp) scaffolds seeded with mesenchymal stem cells (MSCs) and to identify the appropriate reference gene. Methods Ten MSCs-HAp composites were used for RNA isolation by methods based on simplified homogenization steps and column-based purification procedures, while the remaining RNA (n = 13) was extracted by traditional single-step isolation methods. The differences between the two procedures regarding the operation time, RNA quantity and quality were evaluated. Quantitative real-time PCR (qRT-PCR) analysis was performed to identify the appropriate reference gene. Results The simplified method showed significant superiority in operation time (P < 0.001), RNA concentration (P < 0.001), A260/280 ratio (P = 0.005) and A260/230 ratio (P < 0.001). The average integrity number and 28 s/18 s ratio of RNA yielded by the simplified method were 9.1 ± 0.2 and 1.3 ± 0.1, respectively. The qRT-PCR analysis results indicated that the cycle threshold (Ct) values of GAPDH were significantly higher than those of the remaining 2 reference genes (ACTB and RPL13A) in the RNA samples obtained by the simplified and traditional methods (P < 0.05). The standard deviations of the ΔCt value (the difference between the Ct value and the minimum) of ACTB were higher than those of GAPDH or RPL13A, regardless of the RNA isolation method. Conclusion The simplified method could extract intact RNA from biofabricating MSCs-HAp scaffolds and was superior to the traditional single-step procedure in operation time, RNA quantity and quality. GAPDH was identified as the most appropriate reference gene in MSCs-HAp scaffold composites due to its high quantity and good stability.