Quantitative Real-Time PCR for Lung Gene Expression

We isolated RNA from the lungs via Triazol (Invitrogen) and treated it with DNase I (Invitrogen). We used random primers (Invitrogen) and Superscript II (Invitrogen) to synthesize first-strand complementary DNAs from equivalent amounts of RNA from each sample. We performed real-time RT-PCR in a 7000 Real-Time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems). Data were generated by the comparative threshold cycle (ΔCT) method by normalizing to HPRT [26 (link)]. Forward and reverse primers amplifying are as follows, respectively:
M2:5′GAGGTCGAAACG CCT 3′ & 5′CTGTTCCTTTCGATATTCTTCCC3′, CYP4F18:5′ AGAGCCTGGTGCGAACCTT 3′ & 5’ TGGAATATGCGGATGACTGG 3’, CYP4F16:5’GGAGTGGCTTCCTGGATTTT3’& 5’ATGCAGGGTCAACAATCCTC3’, TBXAS1:5’AGGCTTCTGAAAGAGGTGGACCT3′ & 5′TGAAATCACCATGTCCAGATAC3′, ALOX5:5′ATGCCCTCCTACACTGTCAC3′ & 5′CCACTCCATCCATCTATACT3′, ALOX5ap:5’CTCCCAGATAGCCGACAAAG3’ & 5’CAGAACTGCGTAGATGCGTA3’, COTL1:5’GATGAGGGCAAACTTGGATCT3’ & 5’GAGCAGATTACCAGCACTTCA3’, GGGT1: 5’AGGAGAGACGGTGACT3' & 5' GGCATAGGCAAACCGA3', DPEP2: 5’CTGACCTTTCTCTGCCACA3’ &5’GAATCTTCCTGATGACCTCCTG3’