We collected serum samples from all subjects during the summer months (June–August) in 2017 to avoid seasonal bias from variable sunlight exposure. We collected all samples after an overnight fast, in the morning between 8 and 9 a.m. First, we collected venous peripheral blood to normal serum tubes, let them stand and clot in room temperature for 30–60 minutes and then centrifuged them at 2500 x g for 10 minutes. The supernatants were then transferred to 1.5 ml tubes in 250 µl aliquots and stored immediately at -80 °C until analysis.
Growth protocol
We have previously identified four Finnish families with X-linked PLS3 osteoporosis due to different mutations in PLS3 . The first family (Family A) has an intronic splice site mutation c.73–24T>A (p.Asp25Alafs*17) identified by exome sequencing as previously described. The second family (Family B) includes three subjects with an intragenic tandem duplication within PLS3 as identified by genomic hybridization array (array-CGH). The third family (Family C) consists of a single subject with a de novo heterozygous missense mutation c.1424A>G (p.AsnN446Ser ) in exon 12 identified by Sanger sequencing of the whole PLS3 gene as previously described. The fourth family (Family D) includes two subjects with a nonsense mutation c.766C>T (p.Arg256*) in exon 8, also identified by conventional Sanger sequencing of the whole PLS3 gene.
Extracted molecule
total RNA
Extraction protocol
Serum was thawed at room temperature and centrifuged at room temperature for 5 minutes at 12,000 x g in order to remove cellular debris. RNA extraction was performed using precisely 200 µl serum as previously described (8,15) using the miRNeasy Mini Kit (Qiagen, Germany) with the following modification: synthetic, non-human oligonucleotides (Qiagen, Germany) were added to the Qiazol lysis buffer, glycogen was added to the precipitation at a final concentration of 0.5 mg/mL to enhance precipitation, and columns were washed three times with Ethanol before elution in 30 µl nuclease-free water. Eluted total RNA was stored at -80°C until further analysis.
Label
SYBR Green
Label protocol
n/a
Hybridization protocol
n/a
Scan protocol
n/a
Description
case SAMPLE 20
Data processing
Cq-values were called using the second-derivate maximum method. Data quality was assessed using spike-in controls to determine RNA extraction efficiency, enzymatic inhibition, and overall variability. Data normalization was performed using the global mean (i.e. average Cq-value for all endogenous microRNAs with Cq<35) to obtain delta Cq-values. normalized data file contains global-mean normalized delta Cq-value (dCqs) Log2 Fold Change = log2 (PLS3 MP vs MN)
