Computational protocol: Gene expression profiling of peripheral blood cells: new insights into Ewing sarcoma biology and clinical applications

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Protocol publication

[…] Five hundred nanogram of total RNA was reverse-transcribed with oligo(dT)12–18 primers and random hexamers using High-Capacity cDNA Reverse Transcription kit (Life Technologies). cDNA quality was assessed by PCR using GAPDH primers (Table ). All reactions were performed in duplicate or triplicate using the TaqMan Gene Expression Assays (Life Technologies) (Table ) in the 7500 Fast Real-Time PCR System (Life Technologies), according to the manufacturer’s recommendations. Experimental data were analyzed using threshold-cycle (Ct) values generated in the SDS 2.1 software (Life Technologies). Mean expression level of two reference genes PSMC4 and EIF2B1 served as endogenous control. Endogenous controls were selected from a set of 4 candidate genes (EIF2B1, MRPL19, POP4, and PSMC4) based on the most stable expression across different sample types (blood, tumor tissue, and cell line), which was calculated using geNorm [] and NormFinder [] algorithms. Negative controls were also included in every step of the procedure. Total RNA from RD-ES cell line served as an inter-plate calibrator. [...] Chromas Lite 2.01 software (Technelysium Pty, Ltd, South Brisbane, Australia) and BLAST software were used for the analysis of sequencing data (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Relative gene expression levels were calculated using 2−ΔΔCt method []. Statistical analysis was performed using GraphPad QuickCalcs (Grubbs’ test to detect outliers; t test to compare two means between groups; chi-square test for contingency tables analysis) (GraphPad Software, Inc., La Jolla, CA, USA).For the survival analysis, the Kaplan–Meier estimator was used with the log-rank tests for bivariate comparisons using STATISTICA 7.0 software (Statsoft, Tulsa, OK, USA). Overall survival (OS) time for the assessment of prognostic value of clinical and molecular parameters was calculated from the date of start of therapy to the date of the most recent follow-up (censored data) or death. The differences were considered statistically significant if the p values were below 0.05. […]

Pipeline specifications

Software tools NormFinder, Statistica
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Neoplasms, Osteolysis, Sarcoma, Ewing