Megaplex kit

The discovery cohort consisting of four DAWT, eight FHWT, and six non-neoplastic control patient autopsy samples were included. All samples used in this phase were frozen tissue. cDNA was synthesized from 1 μg of total RNA using a Megaplex kit (4444745, Applied Biosystems). To create a miRNA expression profile, 756 miRNAs were divided into cardA v2.0 and B v3.0 and tested following the manufacturer's instructions. TaqMan MicroRNA Master Mix was used for amplification, performed in a Viia 7 thermocycler (Applied Biosystems). MiRNAs with Cq values >35 were considered non-informative and were excluded.
Tumoral Cq values were normalized to those of U6 snRNA and RNU48 endogenous controls and compared with those of non-neoplastic kidney tissues using the relative quantification formula 2^−ΔΔCt. Heatmaps for the relative expression values (fold change) were generated using MultiExperiment Viewer (MeV) v.4.6. Data were evaluated by comparing expression values between DAWT, FHWT, and control samples. The SAM (Significance Analysis of Microarrays) program was used to evaluate significant differences between miRNAs groups. Values >2 were considered to correspond to upregulation, values < −2 to downregulation, and values between −2 and 2 to no change in expression.

It was performed using Applied Biosystems 7900 HT real‐time PCR instrument (Applied Biosystems Inc., Foster City CA, USA) and human miR card A (Applied Biosystems by Life Technologies, NY, USA), containing the most common 365 human miR assays, including those related to liver, in addition to selected small nucleolar RNAs. RNA was converted to cDNA by priming with a mixture of looped primers (MegaPlex kit, Applied Biosystems by Life Technologies) according to manufacturer's instructions. Pre‐amplification was performed using 3 μL of input RNA with PreAmp kit (Life Technologies). 9 μL of pre‐amplified cDNA was used for RT–PCR with the card.

Mature miRNA profiling was performed using an Applied Biosystems 7900 HT real-time PCR instrument and human MicroRNA Array pool A (Applied Biosystems, Foster City, CA, USA), containing 365 different, most common human miRNA assays in addition to selected small nucleolar RNAs (snoRNAs). RNA was converted to cDNA by priming with a mixture of looped primers (MegaPlex kit, Applied Biosystems) in compliance with the manufacturer’s instructions. Preamplification was performed using 3 μL of input RNA with PreAmp kit (Applied Biosystems). 9 μL of preamplified cDNA was used for RT–PCR with MicroRNA Array pool A.