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. 2023 Apr 24;13:6620. doi: 10.1038/s41598-023-33698-8

Figure 1.

Figure 1

Schematic of EBC-microRNA detection and verification: (A, left panels) Exhaled microRNA collection and amplification. The method used the handheld disposable Rtube, typical examples of URT-PCR melt curves for miRs 18a, 200c, 212 are shown. (B, right gel panel): Specificity testing of designed microRNA PCR primers. PCR products were loaded in EtBr gel. Note that 40 bp URTtag + 22 bp microRNA template = 62 bp PCR product. Lane 1: 10 bp DNA ladder; lane 2: cDNA of cultured lung cell microRNA and mRNA (total RNA extract), with both polyadenylation and RT steps; lane 3: No RT step, total RNA from cultured lung cells; lane 4: cDNA of mRNA (no poly-adenylation step, total RNA from cultured lung cells); lane 5: cDNA of EBC microRNA and mRNA (all steps included); lane 6: Genomic DNA spike-in as only template; lane 7: Water blank/no template control. For a microRNA-specific PCR primer set, microRNA-size PCR bands are uniquely seen in lane 2 (cultured lung cell mix RNA extract template, all polyadenylation and RT steps included), and lane 5 (EBC RNA extract template, all polyadenylation and RT steps included). PCR product size is ~ 60–63, reflecting the 20–23 bp microRNA template, and the 40 bp URT tag integrated at the RT step21–23.