Evaluation of RNA from human trabecular bone and identification of stable reference genes. Issue 6 (4th January 2018)

• Record Type: Journal Article
• Authors: Cepollaro, Simona; Della Bella, Elena; de Biase, Dario; Visani, Michela; Fini, Milena
• Is Part Of: Journal of cellular physiology. Volume 233:Issue 6(2018:Jun.)
• Page Start: 4401
• Page End: 4407
• Publication Date: 2018-01-04
• Subjects: gene expression -- geNorm -- human bone -- reference gene stability -- RNA isolation -- RNA quality; Physiology -- Periodicals; Cell physiology -- Periodicals; 571.6
• DOI: 10.1002/jcp.26319
• Languages: English
• ISSNs: 0021-9541

Abstract

Abstract : The isolation of good quality RNA from tissues is an essential prerequisite for gene expression analysis to study pathophysiological processes. This study evaluated the RNA isolated from human trabecular bone and defined a set of stable reference genes. After pulverization, RNA was extracted with a phenol/chloroform method and then purified using silica columns. The A260/280 ratio, A260/230 ratio, RIN, and ribosomal ratio were measured to evaluate RNA quality and integrity. Moreover, the expression of six candidates was analyzed by qPCR and different algorithms were applied to assess reference gene stability. A good purity and quality of RNA was achieved according to A260/280 and A260/230 ratios, and RIN values. TBP, YWHAZ, and PGK1 were the most stable reference genes that should be used for gene expression analysis. In summary, the method proposed is suitable for gene expression evaluation in human bone and a set of reliable reference genes has been identified. Abstract : Good quality RNA was isolated from human trabecular bone for gene expression analysis and the most stable reference genes were selected from a list of candidates. TBP, YWHAZ, and PGK1 were identified as the best reference genes for human trabecular bone. The method proposed is a good starting point for the evaluation of gene expression in real‐time quantitative PCR and microarray.