Polytron aggregate

Mouse L2–3 vertebra bone RNA were extracted using TRI Reagent (MRC Inc) according to the manufacturer's recommendation followed by DNase digestion and column clean‐up using QIAGEN mini columns. Briefly, at RNA isolation from bone tissue at the time of euthanization, the L2–3 vertebra was collected and bone marrow cells were flushed out with Eagle's MEM + Hanks' salts after cleaning the surrounding connective tissue. L2–3 vertebra bone was placed in 1000‐μl TRI Reagent with five metal beads and homogenized using a polytron‐aggregate (Kinematica). Then 100 μl of 1‐bromo‐3‐chloropropane was added and the mixture was centrifuged for 15 min at a speed of 16,000 rpm at 4°C. There was 450 μl of supernatant taken and an equal volume of isopropanol was added and centrifuged for an additional 15 min (16,000 rpm at 4°C). After washing the RNA pellet with 75% ethanol, isolated RNA was resuspended in RNase free water. Reverse transcription was performed using an iScript cDNA synthesis kit from Bio‐Rad. Real‐time RT‐PCR was performed using SYBR Green and an ABI 7500 fast‐sequence detection system (Applied Biosystems). As we described previously,⁽³⁶⁾ the same procedures were used for RNA isolation from ex vivo cultured nonadherent bone marrow cells and attached stromal cells as colony‐forming fibroblasts.

The tissue samples were mixed in TRIzol reagent (Invitrogen, São Paulo, Brazil) for 30 s using specific equipment for this purpose (Polytron-Aggregate, Kinematica, Littau/Luzern, Switzerland) at maximum speed. Subsequently, the homogenates were centrifuged at 6,000 g, and total RNA was isolated according to the manufacturer’s instructions and quantified by spectrophotometry. RNA integrity was assessed by agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized from 2 μg total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA).