Transcriptomic Profiling of Pulmonary Autopsy

RNA was isolate from tissue samples using the Maxwell RNA extraction system (Promega, Madison, Wisconsin). mRNA expression data of pulmonary autopsy specimens was obtained via the nCounter Analysis System (NanoString Technologies, Seattle, WA) using the PanCancer Progression Panel (770 genes including 30 reference genes). Normalization of raw counts was performed using the nSolver analysis software version 3.0 (NanoString Technologies, Seattle, WA) and a modified version of the nCounter advanced analysis module (version 1.1.5). The normalization process included positive normalization (geometric mean), negative normalization (arithmetic mean) and reference normalization (geometric mean) using the 5 most suitable reference genes from the total of 30 available reference genes selected by the geNorm algorithm [73 (link)]. Further analysis of the resulting log2 mRNA counts was performed using custom R code. A Shapiro-Wilks test was performed on all intra-group gene expressions which showed that the vast majority of expression data is normally distributed (>85%, α = 0.05). Raw counts and normalised data for analyzed genes are found in S3 Table.

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Lee A.J., Feng E., Chew M.V., Balint E., Poznanski S.M., Giles E., Zhang A., Marzok A., Revill S.D., Vahedi F., Dubey A., Ayaub E., Jimenez-Saiz R., McGrath J.J., Ritchie T.M., Jordana M., Jonigk D.D., Ackermann M., Ask K., Miller M., Richards C.D, & Ashkar A.A. (2022). Type I interferon regulates proteolysis by macrophages to prevent immunopathology following viral infection. PLoS Pathogens, 18(5), e1010471.

Publication 2022

nCounter Analysis System nSolver analysis software version 3.0

Autopsy Gene expressions Genes Mrna Progression Promega Pulmonary Tissue

Corresponding Organization : Johannes Gutenberg University Mainz

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Variable analysis

independent variables

Tissue samples

dependent variables

MRNA expression data

control variables

Reference genes (30 total, 5 most suitable genes selected by geNorm algorithm)
Positive and negative normalization

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