Mircury lna mirna pcr assay

For measurement of miRNAs, 300 µl of the plasma aliquot was thawed to room temperature. RNA extraction was performed on a safety cabinet using the NucleoSpin^® miRNA Plasma Kit from Macherey–Nagel. To the 300 µl sample plasma, 89.5 µl MLP buffer and 0.5 µl glycogen were first added, homogenized, and then incubated for 3 min at room temperature. The further procedure followed the protocol given by the manufacturer. For RT-qPCR, the miRCURY LNA miRNA PCR assay from Qiagen was used in combination with the qPCR-CYBR master mix from Steinbrenner. First, the cDNA was diluted 1:30 by adding 29 µl of RNAse-free water to 1 µl of cDNA. Then, the reaction mix was prepared, which consisted of the following components: 2.5 µl 2 × qPCR-CYBR Master Mix, 0.5 µl PCR Primer Mix, 0.4 µl RNAse-free H2O 0.4, 1.6 µl 1:30 diluted cDNA 1.6. The PCR plate containing the samples was placed in the qTower3 84G.

A specific miRCURY LNA miRNA PCR Assay (QIAGEN, RRID:SCR_008539, Cod. 339,306-YP00204380) was used to analyze hsa-miR-197-3p concentrations by RT-qPCR and ddPCR. Standard curves were generated for hsa-miR-197-3p using Specific synthetic Locked Nucleic Acid (LNA) oligonucleotides with the following sequence: synthetic hsa-miR-197-3p 5’-rUrUrCrArCrCrArCrCrUrUrCrUrCrCrArCrCrCrArGrC-3’ (QIAGEN, RRID:SCR_008539, Milan, Italy).
Stock solutions (100 μM) of synthetic oligonucleotides, in RNase-free/DNase-free water, were prepared following the manufacturer’s instructions. In order to construct the standard curve of synthetic oligonucleotides miR-197-3p, serial dilutions of the stock solution were performed at 1:10 from 10¹³ copies/μl to 10⁰ copies/μl. The obtained miR-197-3p standard curve was used to quantify miRNAs absolutely by RT-qPCR^{70 (link)} and one point of the curve was used as a positive control in ddPCR.

Total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. RNA quality and quantity were assessed using a spectrophotometric method (NanoDrop 2000, Thermo Scientific, Wilmington, NC, USA). A 10 ng sample of total RNA was reverse transcribed with an miRCURY LNA RT Kit (Qiagen), and the expression of a panel including 21 miRNAs involved in the IGF-1 signaling pathway was evaluated using a miRCURY LNA SYBR Green PCR Kit and a miRCURY LNA miRNA PCR Assay (Qiagen). The 21 miRNAs were the following: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-let-7d-5p, hsa-miR-145-5p, hsa-miR-19a-3p, hsa-miR-195-5p, hsa-miR-126-3p, hsa-miR-150-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-21-5p, hsa-miR-223-3p, hsa-miR-9-5p, hsa-miR-7-5p, hsa-let-7e-5p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-let-7i-5p, and hsa-miR-100-5p. The Ct data were normalized against the geometric mean of three reference miRNAs (U6 snRNA, SNORD38B, and SNORD49A) whose stability in pancreatic normal tissue, pancreatitis, and PDAC has been validated in our samples using the RefFinder algorithm [20 (link)]. The miRNA expression data are presented as 2^−∆Ct values.

Total RNA was extracted with Tri-Reagent^TM (Sigma, St. Louis, MO, United States) and quantified by spectrophotometry (NanoDrop^® ND-1000 UV-Vis Spectrophotometer; NanoDrop Technologies, Wilmington, DE, United States). The mRNA levels of bone morphogenetic protein 2 (BMP2) and cyclin D1 (CycD1) were determined by quantitative real-time RT-PCR (LightCycler^® 96, Roche Diagnostics, Basel, Switzerland). A SensiFAST SYBR, NO-ROX ONE-STEP (Bioline) was used to quantify mRNA expression levels. mRNA expression was expressed as a value normalized to levels of GAPDH mRNA (Table 1).
miRNA-223-3p expression in VSMC was performed by reverse transcription and real-time quantitative RT-PCR (Qiagen). Firstly, 1 μl of RNA eluate was reverse transcribed in 10 μl reaction using the miRCURY LNA RT Kit (Qiagen) according to the manufacturer’s instructions. The cDNA was diluted 30-fold, 3 μl of this diluted cDNA was used as a template and assayed in 10 μl PCR reactions according to the protocols of the miRCURY LNA miRNA PCR Assay (Qiagen). The amplification was performed in a LightCycler 96 Real-Time PCR System (Roche).

For miRNA expression analysis, the total RNA was extracted from cell culture media by using TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse-transcription of RNA was performed using the miRCURY LNA RT Kit according to the manufacturer’s instructions (Qiagen). qRT-PCR assays were carried out in a Rotor-Gene^®Q PCR System (Qiagen) using a miRCURY LNA SYBR^® Green PCR Kit and miRCURY LNA miRNA PCR Assay according to the manufacturer’s instructions (Qiagen). Briefly, each reaction was performed in a final volume of 10 μL containing two μL of the cDNA, a master mix containing 5 μL of 2× miRCURY SYBR Green PCR Master Mix, 1 μL of miRCURY LNA miRNA PCR Assay, and RNase-free water. The amplification profile was: PCR initial heat activation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 10 s and combined annealing/extension at 56 °C for 60 s. The expression of mir-146b and mir-223 was normalized to RNU6B and calculated as 2-^ΔΔCt.

Gene expression was determined by qRT-PCR analysis using QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems), Blank qPCR Master Mix (EURx) and the indicated Taq-Man probes (Applied Biosystems): human: GAPDH (Hs99999905_m1), SNAI1 (Hs00195591_m1) and EZRIN (Hs00931653_m1). The mRNA expression level for all of the samples was normalized to the housekeeping gene GAPDH, using the 2^−ΔCt method.
For the evaluation of miRNA expression by quantitative real-time PCR, SYBR Green qPCR Master Mix (EURx) with LNA™ PCR primer set (Exiqon) or miRCURY LNA miRNA PCR Assay (Qiagen) for human miR-28-3p, miR-193a-5p, miR-218-5p, miR452-5p and miR-103a-3p were used. The miRNAs expression levels were quantified using the 2^−ΔCt method, using miR-103a-3p as a relative control.