RT-qPCR Analysis of miRNA and mRNA

miRNA (100 ng) or mRNA (200 ng) were reverse transcribed to cDNA, using Taqman MicroRNA Reverse Transcription reagent or High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Predeveloped primer/probe sets were purchased from Applied Biosystems (TaqMan miRNA assays or TaqMan gene assays). The quantitative fluorogenic amplification of cDNA (RT-qPCR) was performed using an ABI 7500 Real Time PCR (Applied Biosystems) or Eppendorf RealPlex (Eppendorf) instruments, respectively. RNU44 (based on manufacturer’s recommendations (Applied Biosystem) and previous reports [38 (link), 39 (link)]) or ribosomal RNA (18S) [40 (link), 41 (link)] was used as the reference miRNA or gene, respectively. The cycle threshold (Ct) value obtained was normalized to that of the RNU44 or 18S gene (∆Ct). Data were expressed as 2^-∆∆Ct [42 (link)] where ∆∆Ct was calculated from subtracting individual ∆Ct from ∆Ct of non-asthmatic media control at baseline.