Taqman array human microrna a b cards set v3

MicroRNAs were extracted using the NucleoSpin miRNA kit (Macherey Nagel^®) and were reverse-transcribed using the miScript II RT kit (Qiagen^®). Quantitative PCR reactions were carried out in 384-well plates using a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems/ Thermofischer Scientific^®) with the miScript SYBR Green PCR kit (Qiagen^®) and the assays from Qiagen described in Supplemental Table S1. The HsRNU6-21 miScript Primer Assay was used for the normalization of microRNA expression. Analysis of the expression profiling of 754 microRNAs in hES cell lines and MPCS was carried out on three replicates for each RNA sample according to the recommendation of Applied Biosystems. The assay included RT with specific primers, followed by real-time qPCR using the TaqMan Array Human microRNA A + B Cards set v3.0 and TaqMan universal master mix in an Applied Biosystems 7900 Real-Time PCR System. MicroRNA expression levels were normalized to two different internal control small RNAs (RNU48 and U6 snRNA), obtaining similar results. The comparative threshold cycle method was used to calculate the relative microRNA expression.

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 in S1 Data.

HLSC and FIBRO EVs, derived from three different preparations, were used for the extraction of RNA by a mirVana RNA isolation kit (Ambion), according to the manufacturer’s protocol. miRNA content was analyzed using the qRT-PCR method and the Applied Biosystems TaqMan™ Array Human MicroRNA A + B Cards Set v3.0 (Applied Biosystems), run on QuantStudio 12K Flex (Applied Biosystems). Briefly, 140 nanograms of RNA were reverse transcribed with a Megaplex RT Pools kit (Applied Biosystems), according to the manufacturer’s protocol. TaqMan® PreAmp Master Mix 2X (Applied Biosystems) and specific Megaplex™ PreAmp Primers (10X) (Applied Biosystems) were used to pre-amplify each cDNA sample, which were then loaded onto the TaqMan MicroRNA Array. Raw Ct values, the automatic baseline, threshold and comparison of miRNA expression were analyzed using Expression Suite software (Thermo Fisher Scientific, Waltham, MA). Data were matched with the MSC EV miRNA expression dataset, published in Collino et al.^{30 (link)}.
Pathway enrichment analysis was performed using Funrich V3.1.3 software^{63 (link)}, and data with an adjusted p-value < 0.05 were considered to be statistically significant (Hypergeometric test with Boferroni correction).