Mircury lna universal rt cdna synthesis kit

RNA isolation from 200 μl EDTA plasma was performed using the miRCURY RNA Isolation Kit (Exiqon, Vedbaek, Denmark), according to the manufacturer's instructions. Isolated microRNA samples were stored at −80 °C until use. In the identification cohort, cDNA was synthesized using miRCURY LNA Universal RT cDNA synthesis Kit (Exiqon). Initial microRNA detection screening was performed using the 384-well Serum/Plasma Focus microRNA PCR Panel (V4.0) and the ExiLENT SYBR Green Master Mix (Exiqon), measuring 179 human miRNAs. An RNA spike-in kit (Exiqon) was used to monitor the efficiency of RNA isolation, cDNA synthesis and PCR amplification. Spike-in outlier values were calculated using Grubbs’ outlier test and a visual inspection of spike-in line plots was performed. None of the spike-in deviated beyond the 95% confidence interval (CI), and no distinct abnormalities were observed in the spike-in line plots. In the confirmation cohort, cDNA synthesis was done using qScriptTM microRNA cDNA synthesis kit (Quanta Biosciences, USA). Individual microRNA RT-qPCR were performed using LightCycler 480 SYBR Green I Master (Roche Diagnostics, Switzerland). MicroRNA primer sequences were determined using mirBase.org [16 (link)] (Table S1). Determination of the threshold cycle (C_T) and the melting curve analysis for the microRNAs were done using Lightcycler 480 software (Roche Diagnostics).

miRCURY LNA^™ Universal RT cDNA Synthesis Kit (Exiqon, Denmark) was used for quantitative reverse transcription PCR (RT-PCR) assays as described by the manufacturer. 1 μl of extracted RNA was used for a single RT reaction in a total reaction volume of 20 μl. The cDNA obtained was diluted 1:40 for real-time PCR reactions performed using Applied Biosystems Vii7 Sequence Detection System (Applied biosystems, Waltham, MA) in 10 μl PCR mixtures containing ExiLENT SYBR^® Green master mix (Exiqon, Denmark) and specific miRNA primers from the manufacturer. All samples were performed in triplicate. The results were analyzed with the Applied Biosystems SDS software (Applied biosystems, Waltham, MA) for Ct values and melting curve analyses. Relative expression levels were calculated via the 2 –^ΔΔCt method [28 (link)]. U6 snRNA and cel-miR-39 were used as endogenous and exogenous controls [29 (link)], [30 (link)].

Quantitative real-time RT–PCR analysis was done with an ABI Prism 7500 Sequence Detection System using the miRCURY LNA™ Universal RT cDNA Synthesis Kit (Exiqon). The cDNA was diluted 50X and assayed in 10 µl PCR reactions according to the protocol for the miRCURY LNA™ Universal RT microRNA PCR System (Exiqon A/S); each microRNA was assayed twice by qPCR on the plasma Focus microRNA PCR panel. A no-template control (NTC) of water was purified with the samples and profiled like the samples. Analysis of the data was performed using the relative miRNA expression according to the comparative Ct (ΔΔCt) method using negative metastatic samples as reference. We used the geNorm (37 (link)) or the Normfinder algorithm (38 (link)) to select the best combination of two reference genes. Data from multiples plates were normalized using UniSp3 spike-in (Exiqon) as interplate calibrators.

40 ng of total RNA including miRNA was reverse transcribed using the miRCURY LNA™ Universal RT cDNA Synthesis Kit (Exiqon cat. no. 203301). The cDNA template was then amplified using the microRNA Ready-to-Use PCR, Human Panel I + II (Exiqon) in 384-well plates according to the manufacturer’s instruction. The qPCR reactions were run on a LightCycler 480 System (Life Science Roche) using the thermal-cycling parameters recommended by Exiqon (Denaturation at 95 °C 10 min, 45 amplification cycles at 95 °C 10 s 60 °C 1 min). The amplification curves were analyzed by LightCycler 480 Software (Life Science Roche, cat. no. 04994884001). From the 768 wells, 742 miRNA primer sets were used for miRNA expression profiling, there remaining wells contained interplate calibrator oligonucleotides, spike-in control oligonucleotides, and empty wells. Hsa-miR-423-5p was used for normalization. The relative quantification of miRNA expression levels was performed using delta deltaCp method [33 (link)].

Total RNA was isolated using TRIzol (Invitrogen) according to manufacturer’s instructions. RNA quality and quantity was measured by NanoDrop spectrophotometer (ND-1000, Nanodrop Technologies). The same amount of total RNA (500 μg) per sample was used for the cDNA synthesis with miRCURY LNA™ Universal RT cDNA Synthesis Kit (Exiqon).

To examine the expression levels of the endogenous miR-23c, mR-204-3p, miR-520a-5p, miR-489, miR-874 and miR-513a-3p in undifferentiated AB8/13 cells, total RNA enriched in small RNAs was isolated from the cells using TRIzol® reagent (Ambion). miRNA specific reverse transcription was performed with the miRCURY LNA Universal RT cDNA synthesis kit (Exiqon, Vedbaek, Denmark). The cDNA template was diluted 80x and amplified using SYBR® Green master mix (Exiqon, Vedbaek, Denmark) and LNA microRNA-specific primers on a ViiA 7 Real-Time PCR System (Applied Biosystems). All reactions were performed in triplicates. SNORA66, U6 snRNA and RNU1A1 were used as reference.