Computational protocol: The long noncoding RNA HOTAIR has tissue and cell type-dependent effects on HOX gene expression and phenotype of urothelial cancer cells

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Protocol publication

[…] For microarray analysis we used three independent high quality RNA preparations from VM-CUB1_LV, VM-CUB1_HOTAIR 20, 5637_LV and 5637_HOTAIR 4. Synthesis of cDNA and biotin-labeling of cRNA was performed according to the manufacturers’ protocol (3′ IVT Express Kit; Affymetrix). After fragmentation labelled aRNA was hybridized to Affymetrix PrimeViewTM Human Gene Expression Microarrays for 16 h at 45°C, stained by strepatavidin/phycoerythrin and scanned as described in the manufacturers’ protocol.Data analyses on Affymetrix CEL files were conducted with GeneSpring GX software (Vers. 12.5; Agilent Technologies; see GEO accession number GSE57672 for data and further description) []. Input data pre-processing was concluded by baseline transformation to the median of all samples.After grouping of samples according to their respective experimental condition (5637, HOTAIR vs. empty vector; VM-CUB1, HOTAIR vs. empty vector; three replicates each) a given probeset had to be expressed above background in all three replicates in at least one of two conditions to be further analysed in pairwise comparisons. Cell line-specific differential gene expression was statistically determined by unpaired t-test. Resulting p-values were corrected for multiple testing (Benjamini-Hochberg FDR). The significance threshold was set to p (corr) = 0.01.Affymetrix PrimeView CEL files, published under GEO accession GSE47638 [], were processed using GeneSpring GX as described above including probesets expressed above background in at least one of two conditions to be further analysed in pairwise comparison. A fold change cut off of > two-fold was chosen to define differential expression (GSM1153921 vs. GSM1153920, i.e. KYSE180 cells transfected with HOTAIR vector vs. KYSE180 control cells). For comparison with our own expression data we corrected analogous to the procedure used in ref. 21 (p < 0.01 and at least two-fold change). […]

Pipeline specifications

Software tools GeneSpring GX, PrimeView
Applications Miscellaneous, Gene expression microarray analysis
Organisms Homo sapiens
Diseases Carcinoma, Neoplasms