Droplet Digital PCR for miRNA Expression

ddPCR for analysis of expression level of the miRNAs in BAL samples was performed as described in our previous work [38 (link), 39 (link)]. Briefly, TaqMan™ reaction mix (Applied Biosystems) containing sample cDNA was partitioned into aqueous droplets in oil via the QX100 Droplet Generator (Bio-Rad, Pleasanton, CA, USA), and then transferred to a 96-well PCR plate. A two-step thermocycling protocol (95°C ×10min; 40 cycles of [94°C ×30s, 60°C ×60s], 98°C ×10 min) was undertaken in a Bio-Rad C1000 (Bio-Rad). The PCR plate was loaded on Droplet Reader (Bio-Rad), by which copy number of each miRNA per μl PCR reaction mixture was directly determined. Expression of a targeted miRNA in a given sample was calculated using the same equation as described above. However, instead of using Ct, we used copy number of gene in the equation: expression of a miRNA in a given sample = 2−Δcopy number of gene, where Δcopy number of gene = copy number of gene (targeted miRNA) – copy number of gene (U6).