tissue: blood patient diagnosis: Pancreatic Ductal Adenocarcinoma age: 69 gender: Female smoking status: Former alcohol status: Current baseline ca19-9 (u/ml): 2000 baseline cea (µg/l): 7.2 baseline sii: 201 baseline nlr: 1.2 baseline bilirubin (micromol/l): 6 baseline crp (mg/l): 4.9 disease stage: Metastatic disease total cycles of folfirinox: 4 progression after 4 cycles (recist 1.1): Progressive disease overall survival: 5
Extracted molecule
total RNA
Extraction protocol
The whole-blood samples intended for targeted gene expression analysis were collected in Tempus tubes (Applied Biosystems, Foster City, CA, USA) and subsequently stored at −80 °C. Tempus tubes are designed to contain an RNA-stabilizing reagent, which effectively preserves RNA quality, enabling the measurement of gene expression profiles without the need to isolate peripheral blood mononuclear cells. To extract total RNA from the blood in Tempus tubes, a Tempus Spin RNA Isolation Kit from Thermo Fisher Scientific (Waltham, MA, USA) was employed, following the manufac-turer’s instructions. Subsequently, RNA quality was assessed using an Agilent 2100 Bi-oAnalyzer (Santa Clara, CA, USA). Samples with RNA concentrations below 35 mg/mL were excluded from further analysis. Corrected RNA concentrations were calculated based on the percentage of fragments within the 300–4000 nucleotide range to account for RNA degradation. An nCounter® PanCancer Immune Profiling (IP) Panel and nCounter® Myeloid Innate Immunity (MII) Panel, each consisting of 730 genes and 40 housekeeping genes, were used for NanoString targeted gene expression analysis of the 88 whole-blood samples
Label
n.a.
Label protocol
n.a.
Hybridization protocol
For each sample, a total of 200 ng RNA, in a maximum volume of 7 μL, was subjected to hybridization with the two panels for a duration of 17 h at 65 °C, following the protocol provided by the manufacturer (NanoString Technologies Inc., Seattle, WA, USA).
Scan protocol
The nCounter® FLEX platform was utilized to wash away the unbound probes and to count the genes by scanning 490 fields of view (FOV).
Data processing
Data analysis was performed using the advanced analysis module (version 2.0) of nSolver software (version 4.0, NanoString Technology).
