cDNA Synthesis and Human Cystic Fibrosis RT2 Profiler PCR Array
For cDNA synthesis, 1000 ng/µl total RNA was carried out using RT2 First Strand Kit (Qiagen, Germany). cDNA was then added to the RT2 SYBR Green qPCR Master Mix (Qiagen). 25 µl of each sample was aliquotted on 96-well format RT2 Profiler PCR Arrays (Qiagen). The Human Cystic Fibrosis RT2 Profiler PCR Array which uses qRT-PCR based technique consists of 84 genes involved in CF and CFTR-related genes and pathways (Neutrophil chemotaxis; CCL2, CXCR2, EDN1, IL1B, EDNRA, CXCL8 and ITGB2. Immune and inflammatory response; CXCL1/2/8, TNFRSF1A/11A, ACE, IL1B, IL6/10, TGFB1, TLR2/4/5, DEFB1, NFKBIA, NFKB1, ICAM1. Protein folding; HSP90AA1, HSPA1A, HSPA4/8, HSPH1. Ion transport; SCNN1B/G, KCNE1, SLC9A3R1, ADK, NR4A2. Oxidative stress; MBL2, NOS3, CLU, DUSP1, GCLC, LCN2, MSRA, PTGS2, SFTPB), five housekeeping genes (ACTB, B2M, GAPDH, HPRT1 and RPLP0), one genomic DNA control, three reverse-transcription controls and three positive PCR controls. All steps were done according to the manufacturer’s protocol for the Bio-Rad Iq-5 real-time PCR system.