Unisp6 spike in

Complementary DNA (cDNA) was synthesised using miRCURY LNA RT Kit (Qiagen, MD, USA) following the manufacturer’s protocol and as previously reported.⁵⁰ Custom miRCURY LNA miRNA PCR Assay (Qiagen, MD, USA) was used for RT-qPCR amplification of the selected miRNAs. The use of universal RT makes it possible to use one first-strand cDNA synthesis reaction as the template for multiple miRNA real-time PCR assays. In addition, both the forward and reverse PCR amplification primers are miRNA specific and optimisation with LNA provides exceptional sensitivity, extremely low background, and highly specific assays for better discrimination. miRCURY LNA SYBR® Green PCR Kit was used as a master mix for RT-qPCR amplification. The procedure was followed according to the manufacturer’s protocol and as previously reported,⁵⁰ all samples were tested in duplicate and standard deviations of more than 1.000 between reactions were repeated. Uni sp6 spike in (Qiagen) was used across all samples as an internal quality control for evaluating the efficiency of cDNA synthesis and as an inter-plate calibrator in RT-qPCR. We employed three reference miRNAs to regulate variations, normalise small RNA input, and maintain quality control. Saliva samples with miRNA concentrations below 8 ng/µL and Ct values of reference genes exceeding 35 were excluded from the study.

Immediately after extraction and quantification, 0.8μL of the microRNA samples were retrotranscribed in cDNA using the miRCURY LNA™ RT Kit (Qiagen, Hilden, Germany) in a final volume of 10 μL, according to the manufacturer’s instructions. At this stage, 0.5 μL of UniSp6 spike-in (Qiagen, Hilden, Germany) was added to each sample for the evaluation of the quality of the reverse transcription process.
The cDNA samples were stored at −20 °C until processing.
The cDNA was diluted 30 folds before use and 3 μL was assayed with 7 μL of PCR mix, according to the protocol for the miRCURY LNA™ SYBR Green PCR Kit (Qiagen, Hilden, Germany). The PCR amplification was performed on the Bio-Rad CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). Three technical replicates were performed for each biological sample, and the average Cq values of each triplicate were calculated.