Mircury lnatm universal cdna synthesis kit 2

MiRCURY LNA^TM Universal cDNA synthesis Kit II and miRNA PCR system Pick & Mix (Exiqon, Vedbaek, Denmark) were used for cDNA synthesis and qPCR validation analysis of the selected miRNAs according to the manufacturer’s instructions (see Additional file 1: Table S1 for further purchasing information). Due to the low concentration of genetic material associated to vesicles, 2 μL of undiluted miRNAs were used for retrotranscription at 42 °C for 60 min. A third spike-in miRNA (UniSp6) from the same kit (Exiqon) was used as retrotranscription control. After enzyme inhibition at 95 °C for 5 min, cDNA was diluted 1:80 and 4 μL were used for qPCR reaction with ExiLENT SYBR Green Master Mix (Exiqon, Vedbaek, Denmark) on a LightCycler 480 (Roche, Basel, Switzerland), following kit’s instructions. For each sample, miRNAs were analysed in duplicate and the mean value was used in next data analyses. Amplification of the used spike-ins (UniSp4, UniSp5, and UniSp6) was performed in the same PCR pre-designed panels (Exiqon, Vedbaek, Denmark) with an interplate calibrator control miRNA (UniSp3).

MiRNA analyses were performed using miRCURY LNA^TM Universal RT microRNA PCR kit (Exiqon, A/S, Vedbaek, DK). Briefly, 50 ng of RNA were reverse transcribed in 10 µl final volume, according to manufacturer’s instructions. Quantitative real-time PCRs were performed in final volumes of 10 µl reaction containing 4 µl 1/25 diluted cDNA template, using SYBR green and LNA^TM enhanced primers (targets: hsa-miR-146a-5p, hsa-miR-155–5p) designed by Exiqon for optimal performance with miRCURY LNA^TM Universal cDNA Synthesis kit II and ExiLENT SYBR. Melting curves were done to confirm that any product was specific to the desired amplicon. Stably expressed miR-103a-3p was employed as reference gene and used for normalizing target expressions. RNA spike-ins and the matching primer pairs, included in the kit, were employed in some experiments as controls of the RNA isolation, the cDNA synthesis reaction and the PCR.

The total RNA isolated from each tissue sample was reverse-transcribed using miRcury LNA^TM Universal cDNA Synthesis kit II (Exiqon, Vedbaek, Denmark), containing 2 μl of Reaction Buffer 5X, 1 μl of Enzyme mix, 10 ng of RNA in a reaction volume of 10 μl, according to the manufacturer’s protocol (Exiqon: Cat. No. 203301, Version 6.1, 04/2015). The reaction conditions were: incubation at 42°C for 60 min, heat-inactivation of the reverse transcriptase at 95°C for 5 min, cooling and storage at 4°C. The retrotranscription was realized by adding a poly-A tail to the mature miR template and synthesizing the cDNA by a poly-T primer with a 3′ degenerate anchor and a 5′ universal tag.