GSM9745856: Plasma, Not Frail, Subject8b; Homo sapiens; miRNA-Seq
Total RNA, including the small RNA fraction, was extracted from 800 ul of plasma EDTA using the miRNeasy Serum/Plasma Advanced Kit (QIAGEN, Hilden, Germany). Briefly, samples were lysed with Buffer RPL to denature proteins and inhibit RNases. Inhibitors were precipitated using Buffer RPP, followed by centrifugation to recover the RNA-containing supernatant. Binding conditions were optimized with isopropanol and the mixture was processed through RNeasy UCP MinElute spin columns. After sequential washes with Buffer RWT, Buffer RPE and 80% ethanol to ensure high purity, the total RNA was eluted in 15 µl of nuclease-free water. NGS libraries were prepared using the QIAseq miRNA Library Kit (QIAGEN, Hilden, Germany). For each sample, the isolated RNA was used as starting material. The library preparation workflow involved the sequential ligation of specialized 3' and 5' adapters to the miRNAs. Following ligation, reverse transcription was performed to synthesize cDNA. During this step, Unique Molecular Indices (UMIs) were integrated into the cDNA molecules to enable unbiased quantification of miRNA expression and minimize PCR-induced bias. The resulting cDNA was purified using QIAseq miRNA NGS beads (QMN beads) and eluted in nuclease-free water.  The purified cDNA was then amplified via PCR using a universal forward primer and sample-specific indexing reverse primers (QIAseq miRNA NGS Index IL) to allow for multiplexing. The final libraries were purified again using a bead-based cleanup procedure. The quality and size distribution of the final miRNA libraries were assessed using the Agilent 2100 Bioanalyzer with a High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA). A typical miRNA-sized library was identified by a peak at approximately 180 bp.