Computational protocol: Differential Inductive Signaling of CD90+ Prostate Cancer-Associated Fibroblasts Compared to Normal Tissue Stromal Mesenchyme Cells

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Protocol publication

[…] A probabilistic comparative analysis between transcriptomes of treated NCCIT was used to highlight differentially expressed genes with respect to that of untreated NCCIT []. Gene expression level was defined as the normalized and summarized intensities of each GeneChip probeset, and was presented as its logarithmic value: X = log2(Normalized intensity). This step was carried out using the standard robust multi-array average (RMA) method [], implemented in the in-house analysis pipeline SBEAMS []. Data were presented on a grayscale indicating RMA-normalized Affymetrix signal intensity []. Signals of 10 or less were represented as white and signals greater than or equal to 10,000 as black. Higher Affymetrix signal (more black) indicated higher levels of gene expression.The strength of differential expression between any pair of experiments was estimated by , where 0 h represented the untreated NCCIT and 3d represented treated NCCIT at 3 d. Reliability of the differential expression was estimated by calculating the probability P = P(X3d > X0h) or P = P(X3d < X0h) according to a statistical model that assumed a normal distribution Xj ∼ N(mj,sj), where mj and sj were the mean and maximum difference, respectively, among the replicates of group j. Consistently, P = P(X3d > X0h) or P = P(X3d < X0h) was reported if m3d > m0h or m3d < m0h. Functional and ontology enrichment analysis was performed using the DAVID web-based tool []. Freely available prediction software for determination of signal peptides and likely cell membrane-spanning sequences was also used. Signal peptides were predicted using SignalP 3.0 [], and transmembrane (TM) regions were predicted using TMHMM 2.0 [] for protein topology and the number of TM helices. Information from both SignalP and TMHMM were combined to identify proteins that contained predicted cleavable signal peptides and no predicted TM segments as reported previously []. […]

Pipeline specifications

Software tools SBEAMS, DAVID, SignalP, TMHMM
Application Membrane protein analysis
Organisms Homo sapiens
Diseases Carcinoma, Neoplasms, Prostatic Neoplasms