Table 1

 Primers and probes for real time quantitative RT-PCR

GenePrimers and probesSequenceProduct size (bp)GenBank accession no
bp, base pairs.
For MUC5AC, reverse transcription of RNA to generate cDNA was performed with Taqman RT reagents (ref. N808-0234; Applied Biosystems, NJ, USA) and the PCR was performed with TaqMan Universal PCR Master Mix (ref. 4304437; Applied Biosystems). The specificity of PCR primers was tested under normal PCR conditions and the products of the reaction were electrophoresed into a 2.5% Nusieve® GTG® agarose gel (BMA, Rockland, ME, USA). One single band with the expected molecular size was observed for MUC5AC and GAPDH. For the validation of the ΔΔCt method, the Ct values for target (MUC5AC) and reference (GAPDH) genes were measured at different input amounts of total RNA (2.34–300 ng); ΔCt values (target v reference) were then plotted against log total RNA and the absolute value of the slope was found to be 0.008 (i.e. <0.1), indicating similar efficiency of the two systems.
MUC5ACForward5′-TGTTCTATGAGGGCTGCGTCT-3′
Reverse5′-ATGTCGTGGGACGCACAGA-3′102U06711
TaqMan probe5′-TGACCGGTGCCACATGACGGA-3′
GAPDHForward5′-AGTGGATATTGTTGCCATCA-3′
Reverse5′-GAAGATGGTGATGGGATTTC-3′146BCO14085
TaqMan probe5′-CAAGCTTCCCGTTCTCAGCC-3′