Taqman human microrna array

In the ‘discovery set’, a global microRNA expression profiling was performed in lymph nodes from six FFPE oral tongue SCC samples (four with macrometastases and two with non-metastatic nodes) using TaqMan Human MicroRNA Arrays (Applied Biosystems, Foster City, CA, USA).
Total RNA (40 ng) from each lymph node sample were reverse transcribed into cDNA using the TaqMan microRNA Kit and Megaplex RT Primers (both from Applied Biosystems). After synthesis, the cDNA was pre-amplified using the TaqMan PreAmp Master Mix Kit and Megaplex PreAmp Primers (Applied Biosystems). The amplified-cDNA was then transferred to the TaqMan Human MicroRNA Array plates and the amplification was carried out in an Applied Biosystems 7900HT Real-Time PCR system.
The data obtained was analyzed using the software DataAssist v3.0 (Applied Biosystems). The fold-change difference between metastatic and non-metastatic lymph node samples was calculated using the 2^-ΔΔCt method [39 (link)]. The small nuclear RNA U6 was used as an endogenous control and the non-metastatic group was assigned as a reference since this was the most stable control in the assay.

Due to limited RNA could be obtained using microdissection technique, a preamplification step was added per manufacture's protocol when miRNA array was performed in two paired samples of ET and NE from same patients for gross screening. The RNA was reverse transcribed using the TaqMan MiRNA Reverse Transcription Kit and the TaqMan miRNA Multiplex RT Assays, Human pool A, B (V2.1, V3.0, respectively). The expression was profiled with TaqMan Human microRNA arrays (V2.1 for pool A and V3.0 for pool B), using the manufacturer's recommended protocol (Applied Biosystems, Foster City, CA, USA).

RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems). cDNA was preamplified using TaqMan PreAmp Master Mix (Applied Biosystems). qRT-PCR was performed with an Applied Biosystem 7900HT thermal cycler using TaqMan human microRNA array (TaqMan Human microRNA Array A #4398977 and B v3.0 #442812; Applied Biosystems) according to manufacturer's instructions. The downstream analysis filtered out miRNAs not detected in both cell lines (293), whereas those specifically expressed either in LAN-5 or in SH-SY5Y were considered and reported as cell line-specific. Data were then normalized calculating the ΔCt value for single miRNA against the average of the specific controls for each card according to manufacturer's instructions. Differential expression analysis was performed according to ΔΔCt method and only RQ ≥ 2 fold-change were considered for further analysis. miRNAs clusters were generated through the DIANA web tool mirPath v2.0 using miRBase MIMAT IDs (Release 21) remapped to the newest human genome assembly (GRCh38) to avoid duplicate entries present in the previous release. Uniquely targets reported in TarBase database v7.0 were included in the clustering, predicted targets were not taken into account. False Discovery Rate (FDR) correction was applied to the original p-value and only clusters with corrected p-values < 0.05 were shown.