An Improved Method for Isolating High-Quality RNA From Rat Bone at Room Temperature Without the Need for Specialized Equipment

Research on bone diseases often requires investigation of bone gene expression. Isolating high-quality RNA is essential to obtain reliable and accurate gene expression data. In an effort to analyze the expression of genes related to osteoporosis in rat bones, we developed an improved method for extraction of high-quality RNA without the need for liquid nitrogen or specialized equipment. This method involved transitioning frozen bone tissues to a more pliable state with RNAlater ice and pulverization of the samples with a simple homogenizer, followed by a phenol-chloroform-based RNA extraction. Spectrophotometric analysis indicated high purity of the isolated RNA. Electrophoresis on agarose gel revealed two well-defined ribosomal RNA bands. Herein, we present a method that consistently yields RNA of high purity and integrity from rat bone.


Introduction
Research on bone diseases, including osteoporosis, metabolic bone disorders, and inherited syndromes, often requires investigation of bone gene expression. The similarities between human and rodent skeleton have made the laboratory rat a valuable model in bone disease research. Obtaining high-quality RNA from bone remains challenging due to the hypocellular, mineralized nature of the tissue.
Tissue homogenization is the first step in RNA isolation. The conventional approach involves grinding frozen bone samples wrapped in foil using a mortar and pestle, pre-chilled with liquid nitrogen [1][2][3][4]. Although grinding with a mortar and pestle is a simple and relatively inexpensive technique, it may be inconvenient, especially when multiple samples need to be processed. Another limitation of the technique is the potential sample loss during the grinding process that may lead to poor RNA yields. Finally, any delay in this method may result in sample thawing and possible RNA degradation.
Several approaches have been proposed as alternatives to the conventional pestle and mortar homogenization of bone tissues. Carter et al. developed a one-way method for RNA isolation using the Bullet Blender Centrifuge Technology (Next Advance, Troy, NY, USA) that produced high-integrity RNA yields [5]. In the same direction, Pedersen et al. [6] opted to pulverize their bone samples with the TissueLyser II instrument (Qiagen, Hilden, Germany) while Kelly et al. [7] employed the Micro-dismembrator (Sartorius Stedim Biotech, Göttingen, Germany) for bone tissue disruption. Despite the satisfactory quantities of high-quality RNA achieved, these techniques demand specialized equipment, which may not be affordable to many research laboratories.
In our laboratory, we conduct research on the effect of various biologic agents on bone mineral density in rats with experimental autoimmune arthritis. We flash-froze tibias and femurs from rats treated with those compounds and stored them at -80°C to analyze the expression of osteoporosis-related genes. Our initial efforts to isolate RNA from those tissues resulted in low yields and, occasionally, highly degraded RNA. Therefore, we developed an improved method that consistently yields high-quality RNA from bone while eliminating the need for liquid nitrogen and specialized equipment. were anesthetized with 5% isoflurane and euthanized by decapitation. Femur and tibia bones were rapidly harvested. The attached muscle tissue was removed with a scalpel and any remaining tissue was rubbed away with a phosphate buffer saline-soaked gauze. Bones were flash-frozen and stored at -80°C prior to RNA isolation.

RNA isolation
Bone samples were transferred from -80°C storage to 2 mL Eppendorf tubes containing 1.5 mL of pre-chilled RNAlater ice (Invitrogen TM , Carlsbad, CA, USA) and then stored at -20°C for at least 16 hours before RNA isolation. Once the initial overnight soak at -20°C was complete, bone samples were removed from RNAlater ice, sectioned into small pieces, weighted, and the desired amount (100 mg) was placed into homogenization tubes containing 1.5 mL of pre-chilled TRIzol Reagent (Invitrogen TM ). The remaining bone tissue was returned to the RNAlater ice Eppendorf tubes and stored at -20°C.
Bone samples were homogenized for a total of 45-60 seconds using a simple laboratory homogenizer. Bone samples preserved in RNAlater ice develop a more rubbery, less brittle, texture that allows disruption with a simple homogenizer. In addition, we dissected the samples into smaller pieces with a commercial nail nipper to facilitate the homogenization process. We chose to use a nail nipper for the dissection process because it is an affordable instrument. However, other instruments, such as bone cutters or forceps, can also be used.

Discussion
In this paper, we present an improved method for RNA isolation from rat bone tissue without the need for liquid nitrogen or specialized equipment. This method was developed in an effort to analyze the expression of genes related to osteoporosis in rat bones. Extracting high-quality RNA is essential to obtain reliable and accurate gene expression data. However, there is a limited number of studies on RNA extraction from bone tissue and they all involve the use of specialized equipment [5][6][7].
Our initial attempts involved grinding bone samples with a pestle and mortar, pre-chilled with liquid nitrogen. Apart from the health and safety hazards associated with liquid nitrogen use, the technique resulted in particularly low RNA yields. We found that bone samples frozen with liquid nitrogen became brittle and typically shattered into small pieces during the grinding process. Fragmented pieces of the samples frequently flied out of the mortar. Also, a significant quantity of the sample was practically lost in the surface of the mortar and could not be recovered. Another possible explanation for the poor RNA yields obtained with this approach is that even though we used a pre-chilled spatula for the collection of the powdered tissue, samples potentially thawed during transfer from the mortar to the Eppendorf tubes containing TRIzol.
We then attempted to homogenize bone samples in TRIzol using a power homogenizer. We had successfully used this method in the past for RNA isolation from soft tissues, such as rat liver and brain. Briefly, we transferred bone samples immediately from -80°C storage to homogenization tubes containing pre-chilled TRIzol. During the homogenization process we submerged the tubes in an ice bath to maintain sample at near freezing temperature. Analysis of those samples by agarose gel electrophoresis indicated significant RNA degradation. We assume that this method failed to produce high-quality RNA from bone tissue, owning to the fact that homogenization of hard tissues requires more time that possibly generates heat that leads to RNA degradation.
In order to avoid RNA degradation caused by heat generation during the homogenization process, we incorporated RNAlater ice in our protocol. RNAlater ice is a reagent for transitioning samples from a frozen to a more pliable state that allows sample thawing with no loss of RNA integrity. RNAlater ice has been tested with soft animal tissues, such as brain, liver, kidney, and spleen, and with frozen cell pellets.
RNAlater ice has not been tested on mineralized tissues, on the rationale that it might not penetrate them adequately. Our experiments confirm that RNAlater ice is, also, effective at protecting hard tissues, such as bone, from RNA degradation during thawing. RNAlater ice eliminates the need to work with liquid nitrogen and even allows to further dissect and weight the tissues before homogenization. We believe that this method achieved RNA yields of high purity and integrity due to the inclusion of RNAlater ice in our protocol.

Conclusions
Many science laboratories, nowadays, struggle to operate with limited resources. Acquiring new equipment is not always feasible for many researchers. Herein, we propose a method for RNA isolation from bone tissue without any specialized equipment. This approach consistently yields RNA of high purity and integrity from rat bone. This method could facilitate gene expression studies related not only to osteoporosis, but also bone tumors, osteoarthritis, rheumatic diseases, and bone metabolic disorders. 23. Repeat steps 20 and 21 specified in "RNA isolation".

RNA Extraction From Rat Bone Tissue Stored at -80°C
24. Air dry the pellet for 10 minutes.
25. Resuspend the pellet in 50 μL of DEPC-treated water and pipette up and down until the pellet is completely dissolved.
26. Incubate the sample in a heat block set at 55°C for 10 minutes.

RNA quantification by spectrophotometry
Shake vigorously the samples once the chloroform is added. Phase inversion may occur if samples are not properly mixed.
Shake vigorously the samples once the isopropanol is added. This step is necessary for the formation of the pellet at the bottom of the tube.
Calibrate the spectrophotometer with DEPC-treated water.
We loaded the agarose gel wells with 0.625, 1.25, 2.5, and 5μg of RNA. We found that the appropriate amount for 18S and 28S band visualization was 1.25 μg of RNA.

Additional Information Disclosures
Human subjects: All authors have confirmed that this study did not involve human participants or tissue. Animal subjects: Directorate of Veterinary Services of the Region of Central Macedonia Issued protocol number 10/6-19:1. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.