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-qPCR70 (link) and one point of the curve was used as a positive control in ddPCR.
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-qPCR70 (link) and one point of the curve was used as a positive control in ddPCR.
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