Multiplexed Gene Expression Profiling of Tumor Samples

Tumor RNA samples (1 ng input) were reverse transcribed to cDNA. Primer-specific regions were then linearly amplified through a multiplexed target enrichment approach using the Low RNA Input Kit (NanoString). We used both the Human chimeric antigen receptor (CAR)-T Panel (770 genes plus 10 internal reference genes) and the Human Immune Profiling panel (730 genes plus 40 internal reference genes), each containing probes for immune-related and housekeeping genes. Reaction products were hybridized to cartridges on the NanoString MAX/FLEX Prep Station, which were then scanned on the Digital Analyzer. Read counts were normalized to panel-specific reference genes that were selected using geNorm (20 (link)). Each panel was normalized independently and then merged results were prepared for downstream analyses in nSolver 4.0 with the Advanced Analysis plugin (version 2.0.134). To account for overlapping panel probes, nSolver uses a scaling method that takes the ratio of geometric means of all common probes between the two CodeSets. This ratio was then multiplied to the individual probe counts for both common and unique probes in the subsequent CodeSet. Counts for common probes were averaged in the merged data, while unique probe counts were adjusted by the ratio factor. The resulting values were used for downstream analyses.

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Schafer C.C., Jiang J., Elsamanoudi S., Nousome D., Young D.Y., Song Y., Sesterhenn I.A., Chesnut G.T, & Tan S.H. (2023). Immunologic Assessment of Tumors from a Race-matched Military Cohort Identifies Mast Cell Depletion as a Marker of Prostate Cancer Progression. Cancer Research Communications, 3(8), 1423-1434.

Publication 2023

Low RNA Input Kit

Cdna Chimeric antigen receptor Genes Housekeeping genes Human Primer Tumor rna

Corresponding Organization :

Other organizations : Uniformed Services University of the Health Sciences, Henry M. Jackson Foundation, Frederick National Laboratory for Cancer Research, Spring Branch Medical Center, Walter Reed National Military Medical Center

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Variable analysis

independent variables

Tumor RNA samples (1 ng input)

dependent variables

Gene expression levels from the Human chimeric antigen receptor (CAR)-T Panel (770 genes plus 10 internal reference genes) and the Human Immune Profiling panel (730 genes plus 40 internal reference genes)

control variables

Internal reference genes (20) used for normalization

controls

Positive control: Not specified
Negative control: Not specified

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