Paxgene blood mirna isolation kit

All measurements of miR-199a1 and AXL mRNA expression were conducted in duplicate in cord blood samples from 235 participants in the NEST cohort. Total mRNA was isolated from stored PAXgene tubes of cord blood using the PAXgene blood miRNA isolation kit (Qiagen, Valencia, CA). Expression of miR-199a1 was quantified using Origene’s qStar miRNA detection system (Origene, Rockville, MD) with qStar primer pairs specifically designed for the target (miR-199a1 transcript #HP300226) and its corresponding copy number standard (#HK300226). Plasmid DNA containing a cloned fragment of the target gene was used as the qPCR copy number standard. By utilizing this qPCR standard for the miRNA and employing the standard curve qPCR method, we calculated the absolute copy number of miR-199a1 in each sample. To evaluate reproducibility, 10% repeats were included. Additional details of mRNA expression measurements are described in the Additional file 1: Supplemental methods.

Origene’s qStar mRNA detection system (Origene, Rockville, MD) was used in the quantification of AXL mRNA in cord blood in NEST subjects. qPCR primers for the major AXL transcript (#HK228780) and its corresponding copy number standard (#HK201002) were designed by qStar. All measurements of expression were conducted in duplicate in cord blood samples from 235 participants in the NEST cohort. AXL mRNA was isolated from stored PAXgene tubes of cord blood using the PAXgene blood miRNA isolation kit (Qiagen, Valencia, CA). First strand cDNA conversion of mRNA was performed using Origene’s cDNA synthesis kit (#NP100042). qPCR reactions were run with Kappa Sybr Fast qPCR kit (# KK4604; KapaBiosystems, Boston, MA) in the ABI 7900HT thermocycler (Thermofisher). Ten percent repeats were included to evaluate reproducibility.

RNA isolation from peripheral blood was carried out using PAXgene blood miRNA isolation kit (Qiagen, Hilden, Germany). RNA was isolated from stored muscle biopsies after homogenization with the MagNA Lyser (Roche) as extensively described in the Supplementary Materials and Methods, available at Rheumatology online. Amplification of the BcR was carried out as previously described [32 (link), 33 (link)] and further reported in the online Supplementary Materials and Methods, available at Rheumatology online.