qPCR Validation
cDNA was created for L223 and L373 mucus samples using the Promega
Reverse Transcription System (Promega, Madison, WI) with 500 ng of total
RNA as input. Melt curve analysis and agarose gel electrophoresis were
performed to confirm primer specificity. The reactions for qPCR
consisted of 10 μL SsoAdvanced Universal SYBR Green Supermix (Bio-Rad,
Hercules, CA), 0.2 μM forward and reverse primers, and 2 μL of cDNA in a
final volume of 20 μL. Samples were run in triplicate for each gene on a
CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with
the following thermal cycler conditions: 95 °C for 10 min, 40 cycles of
95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. Relative expression
among L223 and L373 samples was determined by the
2−ΔΔCt method (Livak & Schmittgen, 2001) with β-actin
as the normalizing gene. Primer information is listed in Additional File
1: Table S1.