Taqman array human microrna panels a and b

MicroRNA profiling of samples was performed using TaqMan Array Human MicroRNA panels A and B (Life technologies, CA). Each TLDA card detects 384 features including 377 human miRNAs, three endogenous small RNA controls (one of them being in quadruplicate), and a negative control. 754 human miRNAs were quantified in total. Reverse transcription and pre-amplification were performed following the manufacturer’s instructions (Life technologies, CA). Briefly, 3 μL RNAs from serum or 10 to 300 ng RNAs from PBMCs were reverse transcribed using the Megaplex reverse transcription (RT) reaction in a final volume of 7.5 μL using stem-loop primers designed by Life Technologies. 2.5 μL of this cDNA solution were used for a pre-amplification step in a final volume of 25 μL then diluted four times in distilled water DNase/RNase free (Life Technologies Gibco). Nine μL of diluted pre-amplified product added to 900 μL of total mix were used per TLDA card. Real time quantitative PCR was performed with ViiA7 real-time PCR system, and data were collected with the manufacturer’s ViiA™ Software. Gene Expression Suite software (Applied Biosystems, CA) was further used to process the array data. Automatic thresholds were checked individually and corrected when necessary.

MiRNA profiling was performed using TaqMan Array Human MicroRNA panels A and B (Life Technologies, Thermo Fisher Scientific) to analyze 754 human miRNAs. Reverse transcription and pre-amplification were performed following the manufacturer’s instructions (Life Technologies, Thermo Fisher Scientific). Real-time RT-PCR was performed with the Applied Biosystems StepOne Plus PCR system. For each miRNA, the expression level was determined by the equation 2^−ΔΔCT. Data Assist software (v 3.01) (Life Technologies, Thermo Fisher Scientific) was used to process the array data. Results were validated using single assays for TaqMan qRT-PCR (Life Technologies, Thermo Fisher Scientific) for miR-106a (002169), miR-124 (001182), miR-146a (000468), miR-18b (002217) and miR-372 (000560). U6 snRNA (001973) was used as control to normalize data.
Cluster 3.0/TreeView programs were used for hierarchical clustering analysis of miRNA expression to arrange the samples into groups based on their expression levels, as described by Chiang et al., 2011^{63 (link)}. Prior to hierarchical clustering data was log₂ transformed and expression values on all arrays were translated to the same mean. Spearman rank correlation coefficient was used for calculation of distances between miRNAs. The hierarchical clustering tree was inferred from the distances using average linkage method.