An improved method for isolation of RNA from bone
© Carter et al; licensee BioMed Central Ltd. 2012
Received: 24 August 2011
Accepted: 19 January 2012
Published: 19 January 2012
Bone 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.
We 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.
Our 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.
Obtaining intact, high quality RNA is an essential step in analyzing gene expression. This step is particularly challenging in bone, which contains low numbers of cells embedded within a highly mineralized tissue. As the endocrine functions of bone  and the relationship between bone and adipose physiology  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.
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). Femur bones were harvested from five month old male mice that were fed a defined low fat (D12450B, Research Diets, Inc. New Brunswick, NJ) or high fat diet (D12451, Research Diets, Inc) beginning at four weeks of age. Any attached tissue was quickly removed from the bone using a scalpel before the bone was snap frozen in liquid nitrogen. The bone was stored at -80°C for up to two months before isolating the RNA. For RNA isolation, each bone sample was transferred from -80°C storage to liquid nitrogen until it was divided into two equal portions. To divide the bone, the femurs were cut using diagonal pliers (6 inch/solid joint, TopMost) that are available at hardware stores. One half of the bone was added to a microtube (1.5 ml Eppendorf Safe-Lock) that was prechilled by placing the microtube in a rack surrounded by liquid nitrogen. The remaining half of the bone was immediately returned to the liquid nitrogen and then stored at -80°C.
To facilitate processing multiple samples, we used the Bullet Blender (Next Advance) centrifuge technology that homogenizes tissue using bead disruption of the tissue. A four hour incubation of previously isolated liver RNA with RNase-free beads or untreated beads (Next Advance) demonstrated that RNA is not degraded when in contact with the untreated beads (Figure 1A). We then attempted to isolate RNA from bone using the Bullet Blender, which was kept at 4°C in a cold room.
To determine if our approach is applicable to other methods of homogenizing the bone tissue, we repeated the procedure using a Polytron (Kinematic PT 3100, 7 mm tip) to homogenize the tissue (Figure 2B, lane 5, 6). The reagents and bone were handled as described for the Bullet Blender with the exception that beads are not required when using the Polytron. Homogenization in the Polytron was carried out for 45 seconds at full speed in 13 ml round bottom polypropylene tubes (Sarstedt), which were prechilled in a rack surrounded by liquid nitrogen (Figure 1).
We compared this approach to currently used methods that employ separate steps to pulverize the bone and extract the RNA [3–6]. While the one-step approach has the advantage of maintaining the bone and reagents at near-freezing conditions during the homogenization, the currently used methods can be carried out using a simple mortar and pestle rather than the specialized equipment required for the one-step approach. To compare the quality of RNA obtained with the two approaches, we repeated the RNA isolation using a multi-step method that pulverizes the bone independently of the phenol-guanidinium based RNA extraction. We placed the frozen bone in prechilled sterilized foil followed by refreezing the bone and foil briefly in liquid nitrogen. The foil-wrapped bone was placed in a mortar that was prechilled in liquid nitrogen and the bone was pulverized using a pestle until the bone was powdered . The powdered bone tissue was transferred to a microtube for addition of the TriReagent (1 ml) and extraction and isolation of the RNA. The RNA extract was separated from the powdered bone material by centrifugation at 8,600 × g for fifteen seconds at room temperature followed by isolation of the RNA using an RNeasy Mini Kit (Qiagen) at room temperature.
RNA Quality and Yield
RNA Yield and Quality Using a One-Step Approach to Extract RNA from Bone
26.0 -/+ 7.76
2.09 -/+ 0.015
2.17 -/+ 0.163
8.28 -/+ 0.87
3.17 -/+ 3.04
1.91 -/+ 0.155
5.65 -/+ 2.15
In this study, we present a simple method for isolating bone RNA that relies on homogenizing the bone tissue using reagents and materials that are maintained at temperatures near freezing during a single step that combines fragmentation of the bone with extraction of the RNA. This practical and low cost method is readily adaptable to any homogenization equipment that is compatible with cooling the reagents and materials to near freezing. The results demonstrate that a single step approach consistently gives higher yields of intact RNA with RNA Integrity Numbers necessary to support demanding gene expression applications when compared to current methods.
This approach consolidates extraction of RNA from bone into a single step that allows routine isolation of high quality bone RNA from multiple samples without sample loss or possible cross contamination. This approach will facilitate studies designed to investigate changes in bone gene expression by ensuring a high yield of RNA suitable for demanding gene expression applications.
This study was supported by the American Diabetes Association (grant 1-10-BS-55, ZEF) and the National Institutes of Health (R56DK089020-02, ZEF). This project used Genomics Core facilities that are supported in part by COBRE (NIH P20-RR021945) and CNRU (NIH 1P30-DK072476) center grants from the National Institutes of Health. The 3.2 mm diameter stainless blend beads, 1.4 mm diameter stainless blend beads, and 4.8 mm diameter stainless beads were donated by Next Advance.
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