Designed universal reverse primer

The expressions of cell-free miRNAs (i.e., miR-223, miR-155, miR-181b, and miR-126) were quantified by miRNA-specific Universal ProbeLibrary (UPL) probe-based stem-loop RT-qPCR method as we previously described [23 (link),50 (link)]. Briefly, this quantification technique included two steps: (1) miRNAs were transcribed into cDNA via reverse transcription using miRNA-specific stem-loop RT primer (500 nM, Integrated DNA Technologies, Leuven, Belgium) and TaqMan^™ MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and (2) miRNA quantification was performed by RT-qPCR using designed universal reverse primer (100 μM, Sigma-Aldrich, St. Louis, MO, USA), miRNA-specific forward primer (100 μM, Integrated DNA Technologies), and UPL probe #21 (10 μM, Roche Diagnostics) with recombinant Taq DNA polymerase (5 U/μL, Thermo Scientific, Vilnius, Lithuania) and dNTPs (2.5 mM, Thermo Fisher Scientific). All the measurements were run in triplicate on a QuantStudio^™ 12K Flex qPCR instrument (Applied Biosystems). For normalization, the exogenous ‘spike-in’ control cel-miR-39 was measured in all the samples with the same method as above. Primers and qPCR assays were designed by the software developed by Czimmerer et al. [51 (link)], and oligonucleotides that were used in this study are listed in Supplementary Table S1.

The expression of extracellular and intracellular miR-223 was quantified by miRNA specific Universal ProbeLibrary (UPL)-probe based stem-loop RT-qPCR method (Fejes et al., 2017 (link)). The quantification technique included two steps: (1) miRNAs (input total RNA: 10 ng) were transcribed into cDNA via reverse transcription using miRNA-specific stem loop-RT primer (500 nM, Integrated DNA Technologies, Leuven, Belgium) and TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, United States), and (2) miRNA quantification was performed by RT-qPCR using designed universal reverse primer (100 μM, Sigma-Aldrich), miRNA-specific forward primer (100 μM, Integrated DNA Technologies), and UPL probe #21 (10 μM, Roche Diagnostics, Mannheim, Germany) with Taq polymerase (5 U/μL, Thermo Fisher Scientific, Vilnius, Lithuania) and dNTPs (2.5 mM, Thermo Fisher Scientific). The reactions were preincubated at 95°C for 1 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. All the measurements were conducted in triplicate on a QuantStudio 12 K Flex qPCR instrument (Applied Biosystems). For normalization, the small-nucleolar RNU-43 was measured as a reference gene. Primers and qPCR assays were designed by the software developed by Czimmerer et al. (2013) (link). Oligonucleotides of mature miR-223 that were used in this study are listed in Supplementary Table 2.