Quantification of Cldn6 mRNA Expression in Mouse Lungs

Microarray experiments were designed and mRNA analysis was conducted as already described [21 (link)]. In order to specifically assess Cldn6 mRNA expression throughout development, total RNA was isolated from whole mouse lungs at various time points with an Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA) and treated with DNase. Reverse transcription of RNA was performed using the Invitrogen Superscript III First-Strand Synthesis System (Life Technologies, Grand Island, NY) in order to obtain cDNA for PCR. The following primers were synthesized and HPLC purified by Invitrogen Life Technologies: Cldn6 (For-GCA GTC TCT TTT GCA GGC TC and Rev-CCC AAG ATT TGC AGA CCA GT) and GAPDH (For-TAT GTC GTG GAG TCT ACT GGT and Rev-GAG TTG TCA TAT TTC TCG TGG). cDNA amplification and data analysis were performed using Bio Rad iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) and a Bio Rad Single Color Real Time PCR detection system (Bio-Rad Laboratories). Primers were used at a concentration of 75 nM each in 25-μl reactions. Cycle parameters were as follows: 3 min at 95°C for initial denaturation, followed by 40 cycles composed of 1 min at 95°C, 15 sec at 55°C and 15 s at 72°C. Control wells lacking template or RT were included to identify primer-dimer products and to exclude possible contaminants.