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

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 by methods based on homogenization steps column-based purification procedures, while remaining (n = 13) was extracted traditional single-step methods. The differences between two procedures regarding operation time, quantity quality evaluated. Quantitative real-time PCR (qRT-PCR) analysis performed Results showed significant superiority in time (P < 0.001), concentration A260/280 ratio 0.005) A260/230 0.001). average integrity number 28 s/18 s of yielded 9.1 ± 0.2 1.3 0.1, respectively. qRT-PCR results indicated that cycle threshold (Ct) values GAPDH significantly higher than those 2 genes (ACTB RPL13A) samples obtained 0.05). standard deviations ΔCt value (the difference Ct minimum) ACTB or RPL13A, regardless method. Conclusion could extract intact superior procedure quality. identified as most gene scaffold due its high good stability.