For synthesis of cDNAs, 5 μg total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (M-MLV; Promega), with an oligo-dT 15mer (Promega) used as the primer. The reaction mixture for the reverse transcription reaction was composed of 1.5 U/μL of ribonuclease inhibitor (RNasin; Promega), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM Mgcl2, 10 mM dithiothreitol, 500 μM dNTPs, and 10 U/μL of M-MLV reverse transcriptase. A reaction without reverse transcription, which comprised all these reactants except for the reverse transcriptase enzyme, was run with each experiment to rule out the possibility of contaminating genomic DNA amplification in the PCR reaction step.
For PCR amplification, interleukin-8 primers were designed based on the GenBank human IL-8 complete cDNA sequence (Unigene Hs.624; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) and amplified a 481 bp-product (forward, nucleotide [nt] 489–508: 5′-GTGTGGGTCTGTTGTAGGGT-3′, and reverse, nt 951–970: 5′-CTGTGAGGTAAGATGGTGGC-3′). GAPDH primers were generated from the human GAPDH sequence (GenBank accession No. X01677) and amplified a 165-bp product (forward, nt 76–96: 5′-GTCGGAGTCAACGGATTTGGTCGT-3′, and reverse, nt 226–240: 5′-GACGGTGCCATGGAATTTGCCATG-3′). Two microliters of the cDNA obtained by reverse transcription was used with the PCR reaction mixture composed of 50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 10 mM MgCl2, 200 μM dNTPs, and 20 μg/μL primers. Thin-walled PCR reaction tubes were used for the reactions and the assay was performed on a programmable thermominicycler (MJ Research, Watertown, MA), by using one cycle each that comprised two successive denaturation steps at 94°C for 2 minutes and 1 minute, 50°C for 2 minutes, 72°C for 3 minutes with a 2-second increase in extension temperature with every cycle, followed by another 72°C for 5 minutes. This cycle was then repeated 30 times. The amplification products were analyzed by gel electrophoresis in 1% agarose gels and the sizes of the amplicons were verified by comparing them with a 100-bp DNA marker (GibcoBRL). In separate experiments, multiplex RT-PCR for the presence of transcripts IL-1β, TNF-α, IL-6, granulocyte-macrophage–colony-stimulating factor (GM-CSF), TGF-α, GAPDH, and IL-8 was performed using a kit (CytoXpress; Biosource), according to the manufacturer’s instructions. Each PCR experiment was performed at least five times.