Mircury lna mirna sybr green pcr

Plasma, isolated from the whole blood as previously described (Section 2.4), was processed by using the miRNeasy Serum/Plasma Mini Kit (Qiagen, Hilden, Germany) to isolate total RNA, including microRNAs (miRNAs). Retrotranscription was carried out using the miRCURY LNA miRNA RT Kit (Qiagen, Hilden, Germany), and the obtained cDNA was diluted to 1 : 30, immediately before use. Real-time PCR was run on the MiniOpticon CFX 48 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the miRCURY LNA miRNA SYBR Green PCR and specific miRCURY LNA miRNA PCR Assay (Qiagen, Hilden, Germany), as previously reported [57 (link)]. The miRCURY Primer Assay specific for hsa-miR-195-5p (MIMAT0000461), hsa-miR-153-3p (MIMAT0000439), and hsa-miR-93-5p (MIMAT0000093) was purchased from Qiagen (Hilden, Germany).
The relative miRNA expression was calculated using the Ct method and normalized on miR-93-5p. Several pieces of evidence reported high stability of miR-93-5p in biofluids [58 (link)–61 (link)]; thus, miR-93-5p was suggested as a plasmatic reference gene in the manufacturer's handbook. According to this, the expression levels of miR-93-5p in plasma samples of our cohort showed comparable expression levels without significant difference among groups (data not shown) [49 (link)].

Two microliters of total RNA column eluate (miRNeasy Serum/Plasma Kit, Qiagen, Hilden, Germany) or total RNA column eluate (exoRNeasy Midi Kit) was converted into cDNA in accordance with the miRCURY LNA RT Kit (Qiagen, Hilden, Germany) protocol; then, the sample volume was diluted 1:60, and cDNA was amplified in accordance with the miRCURY LNA miRNA SYBR Green PCR (Qiagen, Hilden, Germany) protocol using the miRCURY LNA miRNA PCR Assay (cat. no. 339306) in a StepOnePlus^TM thermocycler (Applied Biosystems, Waltham, MA, USA). The relative expression of miRNA in the sample was determined by the ∆Ct method, using UniSp6 or hsa-let-7a-5p as the reference RNA.