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.