GSM4562241: Normal-Urine-5; Homo sapiens; miRNA-Seq
Blood samples were allowed to clot at RT for 1-3 hours, centrifuged, processed as serum, filtered for cell contamination, and cryopreserved at -80°C . Aliquots of serum or plasma were thawed only once and mixed, before being analyzed for miR profiling by HTG WTA. Urine samples from healthy donors (n=8) and advanced stage melanoma patients' (n=9) were collected and processed for miR NGS detection. Tumor tissues were obtained from elective surgeries performed at SJHC (D.K.). Formalin-fixed and paraffin-embedded (FFPE) tissues were obtained for HTG WTA Libraries were prepared according to Illumina instrument sequencing protocols. Probe-captured miR samples were amplified and indexed via polymerase chain reaction (PCR) using master mix (dNTP 10 mM, 5X Hemo Klentaq® Reaction buffer, Hemo Klentaq® DNA polymerase (New England Biolabs Inc., Ipswich, MA), forward, and reverse primers (HTG Molecular Diagnostics Inc., Tucson, AZ). Following PCR, library cleanup was performed using Ampure® XP beads (Beckman Coulter Inc., Brea, CA). Samples were mixed and incubated with the beads at RT for 5 minutes. The bead-bound libraries were washed twice with 80% ethanol to remove leftover PCR reagents and enzymes. After washing, the samples were dried at RT for 2 minutes to allow residual ethanol to evaporate. The libraries were eluted off the beads with 10 mM Tris-HCl, pH 8.0.
GEO Accession
GSM4562241