Geneglobe web tool

Based on the literature review, specific miRNAs were selected with potentially stable plasma expression (reference miRNAs, e.g., miR-16-5p, miR-103a-3p, miR-191-5p, miR-1228-3p, miR-520d-5p) and target miRNAs (miR-10b-5p, miR-99a-5p, miR-130a-3p, miR-342-3p, miR-484, miR-486-5p, miR-1260a) [10 (link),17 (link),52 (link),60 (link)] with elevated expression in the plasma of cancer patients. The expression level of selected target and reference miRNAs was determined by the miRCURY LNA miRNA PCR system (Qiagen), and the quality of samples was tested by synthetic spike-in oligonucleotides (RNA Spike-in kit, Qiagen). All protocols for miRNA quantification were performed using a Bravo liquid handling station (Agilent, Santa Clara, CA, USA) and run on a LightCycler 480 instrument (Roche Diagnostics GmbH, Mannheim, Germany) with rapid analysis on LC480 instrument software by the second derivative method for Cq calculation. Secondary analysis of Cq values was performed in the GeneGlobe web tool (Qiagen), where isolation and transcription quality controls were evaluated, a stabilisation factor for reference miRNAs was calculated and data were normalised. These factors were taken into account when calculating the change in expression between the control group and the group of breast cancer patients with the corresponding p-value.

RT² extracellular matrix and adhesion molecule PCR array (Qiagen, Hilden, Germany) was used to study the gene expression profiles of 84 extracellular matrix and cell adhesion molecules in SUM149 cells seeded in growth media conditioned by the secretome of HCMV^- and HCMV⁺ TAMs. Briefly, PCR array was performed using AriaMX System (Agilent, CA, USA) in a 25 µL total volume using RT² SYBR Green Master Mix (Qiagen, Hilden, Germany). PCR thermal profile was 95°C initial denaturation for 10 minutes, followed by 40 cycles at 94°C for 15 seconds and 60°C for 1 minute. Data were analyzed using the Qiagen Gene globe web tool (https://geneglobe.qiagen.com/analyze/) after normalization to ACTB, B2M, GAPDH, HPRT1, and RPLP0 housekeeping genes.