Taqman human microrna array a v2

Nine BF samples, sent to the Catania laboratory,were analyzed for the expression of 384 miRNAs by TaqMan Low-Density Array (TLDAs) technology (Panel A) (Applied Biosystem). Because of the low quantity of samples, no procedure of microvesicles purification has been performed (Fig. 1). This highly specific technology amplifies only mature miRNAs^{43 (link)}. According to a previously published protocol, samples were incubated for 1 min at 100 °C to release nucleic acids^{44 (link)}. Every sample was directly reverse transcribed, without prior RNA purification, using TaqMan MicroRNA Reverse Transcription Kit and Megaplex RT Primers, Human Pool A (Applied Biosystems) in a final volume of 7.5 µl. Preamplification of cDNA from the RT reaction product, using MegaplexPreAmp Primers Pool A and TaqManPreAmp Master Mix (2x; Applied Biosystems), was run in a final volume of 25 µl. Preamplified products were loaded onto TLDAs, TaqMan Human MicroRNA Array A v2.0 (Applied Biosystems). Quantitative RT-PCR reactions were performed on a 7900HT Fast Real Time PCR System (Applied Biosystems) as follows: 94.5 °C for 10 min, followed by 40 amplification cycles of 97 °C for 30 sec and 59.7 °C for 1 min (Fig. 1).

Total RNA from small EV CD81⁺ was extracted with Trizol (Invitrogen), according to the manufacturer’s instructions. Before precipitating the RNA with isopropyl alcohol, 20 μg RNase-free glycogen (Invitrogen) was added to the aqueous phase and the samples were stored for 16h at −80°C. RNA pellets were dissolved in RNase-free water and quantified by Qubit (Invitrogen). About 25 ng of total RNA were retrotranscribed and preamplified. Amplified cDNA products were diluted in 75 μL of RNase-free purified water and loaded onto microfluidic cards of the TaqMan Human MicroRNA Array Av2.0 (Applied Biosystems). Quantitative RT-PCR reactions were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) as follows: 94.5 °C for 10 min, followed by 40 amplification cycles of 97°C for 30 sec and 59.7°C for 1 min.

For fluids, miRNA isolation was performed by using Qiagen miRNeasy Mini Kit (Qiagen GmbH), according to the Qiagen Supplementary Protocol for the purification of small RNAs from serum and plasma and finally eluted in a 30 μl volume of RNAse-free water. Oocytes were incubated, after adding water (2 μl), for 1′ at 100°C according to previously published protocols with minor modifications (El Mouatassim et al., 1999 (link); Di Pietro et al., 2008 (link); Battaglia et al., 2016 (link)) in order to release nucleic acids. Samples (3 μl of total RNA from hFF and human MII oocytes), were retrotranscribed and preamplified. Amplified products were loaded onto microfluidic cards of the TaqMan Human MicroRNA Array A v2.0 (Applied Biosystems). To prepare the real time PCR reaction mix, 9 μl of undiluted pre-amplification product was added to 450 μl of 2X TaqMan Universal PCR Master mix, no AmpErase UNG (Applied Biosystems) and nuclease free water was added to a final volume of 900 μl. 100 μl of PCR reaction mix was loaded onto 384-well TaqMan Low Density Human MicroRNA array cards (TLDA). The qRT-PCR reaction was carried out according to the manufacturer’s instructions in a 7900HT Fast Real Time PCR System (Applied Biosystems).