TITLE: A method for isolating RNA from canine bone 
AUTHORS: Rebecca Nance, Payal Agarwal, Maninder Sandey, Dmytro Starenki, Jey Koehler, Abdul Mohin Sajib, Bruce F Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extracting sufficient quantity and quality RNA from bone is essential for downstream application, such as transcriptomic sequencing, to evaluate gene expression. Isolation of RNA from bone presents a unique challenge owing to the hypocellular, brittle and mineralized matrix, which makes homogenizing the tissue difficult and provides little RNA to work with. Removal of contaminating tissue, such as bone marrow and connective tissue, is essential for isolating RNA that is unique to osteoblasts, osteoclasts and osteocytes. This protocol establishes a method to effectively isolate RNA from normal canine bone cells using the second phalanx, without contamination from other tissue types, for downstream transcriptomic analysis.</div><div class = "text-block">This method combines physical manipulation to remove exterior tissue, washing and centrifugation to remove cells and fat within the diaphysis, homogenization using a mortar and pestle on dry ice prior to bead dissociation, followed by acid guanidinium thiocyanate-phenol-chloroform extraction and column purification to yield sufficiant quantity and quality RNA from canine phalanges. The second phalanx was chosen due to its size small enough to fit into a 1.7 mL microfuge tube, but large enough to provide enough RNA. Mean RNA obtained using this protocol was 14.7 ug of RNA per gram of frozen bone.</div></div>

[STEPS]
?. [Bone Preparation]
Remove all exterior soft tissue around disphysis using scissors/scalpel/KimWipes. Use a scalpel to scrape bone along diaphysis exterior to ensure complete removal of periosteum.
**Isolating RNA that is unique to osteoblasts, osteoclasts, and osteocytes is dependent on sufficient removal of these 'contaminating' exterior tissues.
?. [Bone Preparation]
Cut and discard epiphyses using large bolt cutters, being careful to not disturb the tubular structure of the diaphysis.
?. [Bone Preparation]
Place diaphysis in a 1.7 mL microfuge tube and centrifuge at . Liquid in the bottom after centrifugation is the bone marrow/fat contained within the diaphysis.
Centrifuge: 10.000 34, 10 min
?. [Bone Preparation]
Transfer diaphysis to a new 1.7 mL microfuge tube. Add enough 1x PBS (Phosphate Buffered Saline) to cover the bone completely. Centrifuge at .
Centrifuge: 10.000 34, 5 min
?. [Bone Preparation]
Transfer diaphysis to a new 1.7 mL microfuge tube. Add enough 1x PBS to cover the bone completely and centrifuge again at .
Centrifuge: 10.000 34, 5 min
?. [Bone Preparation]
Snap freeze bone in liquid nitrogen and store at  for up to 8 months until RNA extraction.
-80 °C
?. [Bone Homogenization/RNA Extraction]
Prepare two 1.7 mL Eppendorf Safe Lock** microfuge tubes, each containing  and approximately  and place tubes .
[Tri-Reagent]
[of zirconium oxide beads (0.5 mm diameter)]
on ice
**Eppendorf Safe Lock tubes prevent sample leakage during subsequent Bullet Blender step.
?. [Bone Homogenization/RNA Extraction]
Grind bone into a fine powder using a frozen RNase-free steel mortar and pestle** and hammer on top of a bed of dry ice, working quickly to avoid RNase degradation.
**Mortar and Pestle can be prepared RNase-free by wiping with RNase Away, wrapping in aluminum foil, and baking at 350F for 6 hours or overnight. Freeze in   prior to RNA extraction.
?. [Bone Homogenization/RNA Extraction]
Divide the bone powder into the two previously prepared microfuge tubes containing  and .
[Tri-Reagent]
[zirconium beads]
?. [Bone Homogenization/RNA Extraction]
Using a Bullet Blender at speed "5.5", subject samples to 4 rounds of a  in Bullet Blender with a  between rounds (to keep samples cool and minimize degradation).
[spin]
[incubation]
on ice
?. [Bone Homogenization/RNA Extraction]
Incubate samples for  at .
-80 Room temperature
?. [Bone Homogenization/RNA Extraction]
Add  to each tube and vortex thoroughly. Incubate samples for  at .
[bromochloropropane (BCP)]
-80 Room temperature
?. [Bone Homogenization/RNA Extraction]
Centifuge tubes at .
Centrifuge: 20.000 34, 15 min, 4 10
?. [Bone Homogenization/RNA Extraction]
Carefully remove top aqueous layer and add  and 1/10 volume DNase I Reaction Buffer; Incubate  at .
[DNase I ]
-80 Room temperature
?. [Bone Homogenization/RNA Extraction]
Inactivate DNase by adding  and heat for  at .
[of 25 mM EDTA (included)]
65 °C
?. [Bone Homogenization/RNA Extraction]
Add equal volume 70% ethanol and mix well by pipetting.
?. [Bone Homogenization/RNA Extraction]
Apply sample to RNA column from RNeasy Micro Kit placed in a 2 mL collection tube and centrifuge .
Centrifuge: 21.000 34, 1 min
?. [Bone Homogenization/RNA Extraction]
After discarding eluate, add , incubate on the column for  at , centrifuge .
[Buffer RW1]
65 Room temperature
Centrifuge: 21.000 34, 1 min
?. [Bone Homogenization/RNA Extraction]
Discard flow through, add , incubate on the column for  at , and centrifuge .
[Buffer RPE]
65 Room temperature
Centrifuge: 21.000 34, 1 min
?. [Bone Homogenization/RNA Extraction]
Discard flow through, add , incubate on the column for  at , and centrifuge .
[of 80% ethanol]
65 Room temperature
Centrifuge: 21.000 34, 3 min
?. [Bone Homogenization/RNA Extraction]
Discard flow through and replace 2 mL collection tube and centrifuge the columns at .
Centrifuge: 21.000 34, 5 min
?. [Bone Homogenization/RNA Extraction]
Replace collection tube, add  (preheated to ), incubate for  on the column at , and centrifuge .
[of RNase-free water]
65 °C
65 Room temperature
Centrifuge: 21.000 34, 5 min
?. [Bone Homogenization/RNA Extraction]
Concentration and purity can then be determined using a Nanodrop spectrophotometer.