Laser microdissected patient tissue samples were subjected to total RNA extraction using the mirVana microRNA extraction kit (Ambion, Life Technologies, Darmstadt, Germany)
Label
FAM
Label protocol
1µg of total RNA was reverse transcribed using the High Capacity cDNA reverse transcription kit (Applied Biosystems) and TaqMan® Micro-RNA Megaplex RT Human Pool Sets A & B. The arrays were performed according to the manufacturer’s instructions with an Applied Biosystems 7900HT thermocycler under the following conditions: 50oC for 2 min, 94.5oC for 10 min, followed by 40 cycles of 97oC for 30 sec and 59.7oC for 1 min. Raw data were exported using SDS Relative Quantification Software version 2.2.2 (Applied Biosystems) with automatic baseline and threshold settings.
Hybridization protocol
n/a
Scan protocol
n/a
Description
667 human miRNAs from miRBase version v.10
Data processing
The raw Ct values from the 7900HT Fast Real-Time PCR System were processed and analyzed using the Bioconductor package HTqPCR Version 1.12.0 (Dvinge and Bertone, 2009) in the statistical programming environment R. Rank invariant controls were chosen for ΔCt normalization. For the A plates of all samples the arithmetic mean Ct values of MammU6, RNU44, RNU48 were used for normalization, while in B plates the controls MammU6, RNU44, RNU48, RNU24, RNU43 and RNU6B (last three not present on plate A design) were used for normalization. For the relative quantification of miRNA expression a cut-off was set at a CT value of 40. Probes with Ct values of 40 or greater were considered as undetermined. Sample correlation (Pearson’s correlation) was assessed based on normalized Ct values in a heatmap using hierarchical clustering.
