Mini column

Cartilage samples were pulverized using a Retsch MM200 under cryogenic conditions. On average 150 mg of pulverized cartilage was dissolved in 1 ml of Trizol reagent, and mixed vigorously. After addition of 200 µl of chloroform the sample was mixed and centrifuged for 15 minutes (16,000 g). The clear aqueous layer was transferred to a new vial and 1 volume of 70% ethanol/DEPC-treated water was added to precipitate RNA. RNA was collected using Qiagen mini columns according to the manufacturers protocol and quality was assessed using a Bioanalyzer lab-on-a-chip. RNA integrity numbers above 8 were considered suitable for microarray analysis.

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.

RNA was isolated using mini columns (Qiagen) following the instructions of the manufacturer. 500 μg of total RNA was transcribed into cDNA using OneScript^® cDNA synthesis kit (Applied Biological Materials). Primer pairs for measuring the expression of CB1, MAGL and FAAH are described in Table 1, and were designed using Primer-BLAST tool (available on NCBI website) using NCBI Reference Sequence and checked against the genebank to avoid cross-reactivity with other known sequences.

Tissues isolated from wild-type and ob/ob female C57Bl/6 mice were homogenized in Tri-reagent using a ribolyser and total RNA prepared using Qiagen™ minicolumns according to the manufacturer’s instructions. One µg total RNA was reverse-transcribed using avian reverse transcriptase and random priming in a 50 µl reaction. Two µl cDNA was subsequently used per 50 µl PCR reaction as standard. GAPDH was chosen as the housekeeping genes because it showed consistent C_T values in adipose tissue, adipocytes and soleus muscle. Gene expression assays were obtained from Applied Biosystems Assay-on-Demand predesigned and optimized assays.
Subsequently, tissues were isolated from wild-type female C57Bl/6 mice. Total RNA was extracted and RT PCR performed by Real Time PCR using optimized Assay-on-Demand (Applied Biosystems) primers and probes for Kv1.1, 1.2, 1.3 and 1.5, relative to an endogenous GAPDH control (Wargent et al., 2013 (link)). Expression of each potassium channel was calculated relative to expression of GAPDH in each sample and then the expression levels of Kv1.2 and Kv1.5 were expressed relative to Kv1.3 in the same tissue (Wargent et al., 2013 (link)). Multiple Kv channels were not run in the same PCR reaction in order to prevent competition between the cDNA templates for the amplification reactants and to optimize reaction efficiency.

Cell and bone tissue RNA isolation from in vitro cultured cells and in vivo tissues were extracted using TRI Reagent (MRC Inc., Cincinnati, OH, USA) according to the manufacturer's recommendations followed by DNase digestion and column cleanup using QIAGEN mini columns (QIAGEN, Valencia, CA, USA).22 Reverse transcription was carried out using an iScript cDNA synthesis kit from Bio‐Rad (Hercules, CA, USA). All primers for real‐time PCR analysis used in this report were designed using Primer Express software 2.0.0 (Applied Biosystems, Foster City, CA, USA).