Abstract
BackgroundBone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes. However, progress in understanding the role of bone in determining metabolic health is hampered by the well-described difficulty of obtaining high quality RNA from bone for gene expression analysis using the currently available approaches.ResultsWe developed a simple approach to isolate bone RNA that combines pulverizing the bone and the phenol-guanidinium based RNA extraction in a single step while maintaining near-freezing temperatures. This single step method increases the yield of high quality RNA by eight-fold, with RNA integrity numbers ranging from 6.7 to 9.2.ConclusionsOur streamlined approach substantially increases the yield of high-quality RNA from bone tissue while facilitating safe and efficient processing of multiple samples using readily available platforms. The RNA obtained from this method is suitable for use in gene expression analysis in real-time quantitative PCR, microarray, and next generation sequencing applications.
Highlights
Bone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes
Obtaining intact, high quality RNA is an essential step in analyzing gene expression
Extracting RNA from bone in a single step All animal studies using C57BL/6J male mice were performed with approval from the Pennington Biomedical Research Center Institutional Care and Use Committee using mice purchased from Jackson Laboratory (Bar Harbor, ME)
Summary
Bone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes. High quality RNA is an essential step in analyzing gene expression. This step is challenging in bone, which contains low numbers of cells embedded within a highly mineralized tissue. As the endocrine functions of bone [1] and the relationship between bone and adipose physiology [2] becomes increasingly apparent, the need to isolate high quality RNA for gene expression analysis in bone using the current genome-wide sequencing technologies will gain more importance. The powdered bone is transferred to a second container for extraction of the RNA using a phenol-guanidiniumbased reagent. While these approaches support extraction of RNA from bone, the multiple steps introduce
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