Profiling miRNA Expression in Biofluid Samples

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).