Taqman microrna reverse transcription reagent

Total RNAs were extracted using TRIZOL (Invitrogen, USA). For miRNA analysis, the TaqMan microRNA Reverse Transcription reagents and the Universal PCR Master Mix with microRNA real-time PCR primers (Applied Biosystems) were used. Total RNAs (1 μg) were reverse transcribed to cDNAs. For mRNA expression analysis, 1 μg of total RNAs were reverse-transcribed with the TaqMan Reverse Transcription Reagents (Applied Biosystems N808-0234). Gene expression was measured by SYBR green (Applied Biosystems, USA). U6 and GAPDH were used as endogenous control. The primers used were listed in Table 1.Table 1

Sequences of primers used in qRT-PCR

Name	Forward primer (5′-3′)	Reversed primer (5′-3′)
miR-138	AGCUGGUGUUGUGAAUC	GTGCAGGGTCCGAGGT
U6	GTAGTCGGCGAAGGTCTCAC	ACCGTGGATGCAATGCCTAA
DEK	AAAGCCACCTACAGATGAAGAG	TCCTCTCAGTCAAATCACAAGC
GAPDH	GGTTGTCTCCTGCGACTTCA	GGTGGTCCAGGGTTTCTTACTC

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.