Transcriptome profiling identifies NETs-associated biomarkers in osteoporosis with diabetes
Transcriptome profiling identifies NETs-associated biomarkers in osteoporosis with diabetes
Peripheral blood samples were collected from patients who underwent bone mineral density (BMD) testing at the People's Hospital of Gansu Province. The cohort included 15 patients with osteoporosis combined with diabetes and 15 control subjects. Osteoporosis was defined as a spinal BMD T-score ≤ -2.5 SD, while the control group had a T-score ≥ -1.0 SD. Total RNA was isolated and purified from peripheral blood samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quantity and integrity of the extracted total RNA were assessed prior to library construction. The RNA library was prepared following these steps: mRNA with PolyA tails was captured and fragmented. First-strand cDNA was synthesized using reverse transcriptase, followed by second-strand synthesis using E. coli DNA polymerase I (NEB, Cat. No. M0209) and RNase H (NEB, Cat. No. M0297). The double-stranded DNA was then purified, and the fragment size was selected. The UDG enzyme (NEB, Cat. No. M0280) was used for digestion. Finally, PCR amplification was performed to generate sequencing libraries with a fragment size of 300 bp ± 50 bp.