We collected all serum samples during the spring (January-April) in 2017 to avoid bias from natural sunlight. All samples were taken after a 12-hour fast, in the morning between 8 and 9 am. First venous blood was collected into normal serum tubes and left to stand in room temperature for 30-60 minutes to allow clotting. To separate the serum, the tubes were then centrifuged at 2500 x g for 10 minutes in room temperature. The supernatant was then transferred to 250 µl aliquots in 1.5 ml tubes. The serum was then immediately stored at -80°C.
Extracted molecule
total RNA
Extraction protocol
We isolated RNA was isolated using the miRNeasy Mini Kit (Qiagen, Germany). Serum samples were thawed on ice and centrifuged at 12,000g for 5 minutes to remove any cellular debris. For each sample, 200 µL of serum was mixed with 1000 µL Qiazol and 1µL synthetic Spike-Ins (Exiqon, Denmark). After a 10-minute incubation at room temperature, 200 µL chloroform were added to the lysates followed by cooled centrifugation at 12,000g for 15 minutes at 4°C. Precisely 650 µL of the upper aqueous phase were mixed with 7 µL glycogen (50mg/mL) to enhance precipitation. Samples were transferred to a miRNeasy mini column, RNA was precipitated with 750 µL ethanol followed by washing with RPE and RWT buffer. RNA as eluted in 30 µL nuclease free water and stored at -80°C until further analysis.
Label
SYBR Green
Label protocol
Starting from total RNA samples, we synthesized cDNA using the Universal cDNA Synthesis Kit II (Exiqon, Denmark). We chose reaction conditions according to recommendations by the manufacturer. The protocol was modified in that 4µL of total RNA were used per 10 µl reverse transcription (RT) reaction. To monitor RT efficiency and presence of impurities with inhibitory activity, a synthetic RNA spike-in (cel-miR-39-3p) was added to the RT reaction. PCR amplification was performed in a 384-well plate format using custom pick&mix plates (Exiqon, Denmark) in a Roche LC480 II instrument (Roche, Germany) and EXiLENT SYBR Green mastermix (Exiqon, Denmark) with the following settings: 95°C for 10 min, 45 cycles of 95°C for 10 s and 60°C for 60s, followed by melting curve analysis. To calculate the cycle of quantification values (Cq-values), the second derivative method was used.
Hybridization protocol
n/a
Scan protocol
n/a
Description
CASE
Data processing
Cq-values were normalized to the mean Cq-value in each sample (global mean normalization) by subtracting the individual miRNA Cq-value from the Cq average calculated for that sample.
Altered MicroRNA Profile in Osteoporosis Caused by Impaired WNT signaling
Data table header descriptions
ID_REF
VALUE
Global mean normalized deltaCq (dCq) values are shown. Negative dCq values for a microRNA indicate serum concentrations below the average, positive dCq values indicate serum concentrations above the average.
