Lna based mirna qpcr platform

For miRNA quantitative RT-PCR assay, we used the Locked Nucleic Acid (LNA)-based miRNA qPCR platform from Exiqon: hsa-miR-16-5p, hsa-miR-23a-3p, hsa-miR-92b-3p, hsa-miR-103a-3p, hsa-miR-122-5p, hsa-miR-192-5p, hsa-miR-223-3p, hsa-miR-346, hsa-miR-451a, hsa-miR-619-5p, hsa-miR-1246, hsa-miR-1290, hsa-miR-4704-3p, hsa-miR-4732-5p, hsa-miR-6765-3p and hsa-miR-6778-5p. Total RNA was used for cDNA synthesis using MiRCURY LNA Universal cDNA Synthesis Kit II (Exiqon). The cDNA was diluted and qRT-PCR was carried out using specific, pre-defined microRNA primer pairs and the ExiLENT SYBR Green Master Mix (Exiqon) using an ABI7300 instrument (Applied Biosystems). Two miRNAs (hsa-miR-194-5p and ath-miR-159a) were quantified using TaqMan MicroRNA Assay Kit. All steps were performed following the manufacturer’s protocol. To identify the differentially expressed miRNAs, the expression levels were calculated with Delta Ct method (Delta Ct=Ct _test-Ct_ath-miR-159a).

To validate the sequencing data, 3 miRNAs were subjected to quantitative Real Time-PCR using the Locked Nucleic Acid (LNA™)-based miRNA qPCR platform from Exiqon. Briefly, 5 ng of RNA were used for cDNA synthesis using MiRCURY LNA™ Universal RT kit microRNA PCR. Real time PCR was carried out using specific, pre-defined miRNA primer pairs and the ExiLENT SYBR Green Master Mix (Exiqon), following the manufacturer’s protocol and an ABI7500 instrument (Life Technologies). The expression data was normalized to the small RNA SNORD48 expression levels (Exiqon 203903) and for relative quantification we used the comparative ∆Ct method [46 (link)].

Plasma was separated from 5 ml of EDTA whole blood using a centrifugation step of 3600 rpm for 10 min. The miRNA population was isolated from 200 μl of plasma using the miRCURY RNA Isolation Kit – Biofluids (Exiqon), following the instructions provided in the manual. For the identification of miRNAs we used the Locked Nucleic Acid (LNA™)-based miRNA qPCR platform from Exiqon. Briefly, 4 μl of RNA were used for cDNA synthesis using MiRCURY LNA™ Universal RT kit microRNA PCR. The cDNA was diluted following the manufacturer’s protocol and real time PCR was carried out using specific, pre-defined microRNA primer pairs and the ExiLENT SYBR Green Master Mix (Exiqon), following the manufacturer’s protocol, using a ABI7500 instrument (Lifetechnologies). MiRNAs evaluated in plasma were those identified as differentially expressed between N+ and N0 tumors following small RNA sequencing in this study but which had been previously detected in plasma by a large scale study [38 (link)]: miR-20b (Exiqon 204755), miR-30a (Exiqon 205695), miR-31 (Exiqon 204236), miR-106a (Exiqon 204563), miR-130b (Exiqon 204317), miR-139 (Exiqon 205874), miR-296 (Exiqon 204436), miR-301 (Exiqon 204687), miR-335 (Exiqon 204151), miR-551b (Exiqon 204067). As a reference gene for data normalization we used miR-93 (Exiqon 204715), as suggested by the manufacturer and validated in our samples as stably expressed.