MicroRNA profiles were determined in pre-transplant renal biopsies using micro-fluidic cards comprising of 754 independent microRNAs (TaqMan® Array Human MicroRNA A+B Cards Set v3.0, Applied Biosystems). 10 renal biopsies were used for profiling studies (5 good performing allografts and 5 poor performing allografts).
1 μg of total RNA for each card in the set was reverse transcribed using Megaplex™ Primer Pools, Human Pools Set v3.0 and TaqMan® MicroRNA Reverse Transcription Kit according to the manufacturer’s instructions (Applied Biosystems). QPCR was carried out on 7900HT Real-Time PCR system (Applied Biosystems) using thermal profiles recommended by the manufacturer. Amplification profiles for each sample and target were analysed individually and profiles with Ct above 35, bad passive reference signal, noise spike and high noise were removed from further analyses. Data analysis was performed using RQ Manager 1.2 and RealTime®StatMiner (Integromics). Data were normalised against the following endogenous controls: RNU44, RNU48 and MammU6 or U6snoRNA depending on the card used. The comparative threshold cycle method (ΔΔCT) was used to quantify relative gene expression, and the obtained quantification was transformed to exponential value 2-ΔΔCT [14 (link)]. MicroRNA expression profile generation for validation cohort is described inS1 Data .
1 μg of total RNA for each card in the set was reverse transcribed using Megaplex™ Primer Pools, Human Pools Set v3.0 and TaqMan® MicroRNA Reverse Transcription Kit according to the manufacturer’s instructions (Applied Biosystems). QPCR was carried out on 7900HT Real-Time PCR system (Applied Biosystems) using thermal profiles recommended by the manufacturer. Amplification profiles for each sample and target were analysed individually and profiles with Ct above 35, bad passive reference signal, noise spike and high noise were removed from further analyses. Data analysis was performed using RQ Manager 1.2 and RealTime®StatMiner (Integromics). Data were normalised against the following endogenous controls: RNU44, RNU48 and MammU6 or U6snoRNA depending on the card used. The comparative threshold cycle method (ΔΔCT) was used to quantify relative gene expression, and the obtained quantification was transformed to exponential value 2-ΔΔCT [14 (link)]. MicroRNA expression profile generation for validation cohort is described in
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