Computational protocol: A cross comparison of technologies for the detection of microRNAs in clinical FFPE samples of hepatoblastoma patients

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Protocol publication

[…] Four hundred nanograms of RNA per sample were labelled without amplification using the Affymetrix FlashTag™ Biotin HSR RNA Labeling Kit. The hybridization cocktails were prepared using the Affymetrix Hybridization Wash and Stain Kit and the spike in controls were added from the Eukaryotic Hybridization Control kit. The entire labelled sample was then hybridized on an Affymetrix® GeneChip® miRNA 3.0 array for 16–18 hours at 48 °C in a rotating oven. The microarrays were washed and stained on the fluidics station 450 and scanned in a 3000 7G scanner. The scanned image’s features were converted to numerical values of the probe signal intensity with the Affymetrix GeneChip Command Console software and stored as a CEL file.CEL files were loaded into the statistical computing environment of R and the Oligo package from bioconductor was used to perform normalization of the raw probe intensities using a Robust Multi-Array Average (RMA) approach. The arrays were perfect match only (Pm-only); unmodified Pm intensity values of the probes were used to calculate the median value. As a consequence, the median values of each of the probesets represent the summarized expression values of the transcripts for a particular chip. The threshold for these assays was calculated using the spike-in control with the lowest concentration for each sample. This value is the limit of detection for the microarrays. Any miRNAs below this value were considered not confidently identified (i.e., not beyond noise level) and were removed from further analysis.Quality of the microarray data was assessed using a miRNAv3 Array QC report conducted in R with bioconducter packages oligo and Affy to create plots for the distribution of probe intensity, spike-in controls, and array comparison. These plots are designed to highlight any batch effects that have influenced the array data. Boxplots of probe intensities and histograms of log-intensities were generated to compare probe intensity across the arrays. The probe intensity should be extremely similar across all the arrays in these plots. Spike in control plots assess the success of the poly-A tailing and ligation step in the protocol and confirm the lack of RNAses present in the sample. Nine internal “Bio” controls were also added to the array, which provide feedback on the quality of the hybridization, washing and staining procedures performed. The concentration of these nine controls is sequential with BioB being the lowest concentration. It therefore is at the limit of detection for these arrays and it should be present approximately 70% of the time. Lastly, a comparison of the arrays was measured with an MA plot. The MA plots shows the RMA normalised data of all of the possible comparisons between the pairwise log2 intensities of the samples. […]

Pipeline specifications

Software tools Oligo package, affy
Application Gene expression microarray analysis
Organisms Homo sapiens
Diseases Neoplasms, Hepatoblastoma
Chemicals Formaldehyde