Dnase digest

The mRNA coding for IL-10, IL-1β, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was quantified by quantitative PCR. Total RNA was isolated by phenol chloroform extraction, followed by silica membrane purification and DNAse digest (Qiagen, USA). Thereafter, 1 mg of total RNA from each sample was used for reverse transcription using the Transcriptor first-strand cDNA synthesis kit (Roche) to generate first-strand complementary DNA. A polymerase chain reaction mixture was prepared with the use of SYBR Green I PCR Master Mix (Roche, USA) and followed by amplification on a Roche LightCycler 480 instrument. The primer sequences used were as follows: GAPDH forward 5′-TTCACCACCATGGAGAAGGC-3′ and reverse 5′-GGCATGGACTGTGGTCATGA-3′, IL-1β forward 5′-CAACCAACAAGTGATATTCTCC-3′ and reverse 5′-GATCCACACTCTCCAGCTGCA-3′, IL-10 forward 5′-GGTTGCCAAGCCTTATCGGA-3′ and reverse 5′-ACCTGCTCCACTGCCTTGCT-3′, and TNF-α forward 5′-GCACCACCATCAAGGACTCA-3′ and reverse 5′-TCGGAGGCTCCAGTGAATTCG-3′. Thermal cycling conditions were 10 min at 95°C followed by 45 cycles at 95°C for 10 seconds, 60°C for 20 seconds and 72°C for 20 seconds. Cm values ranged from 25–35 for the cytokine gene transcripts. The expression of each gene was normalized to GAPDH mRNA, and calculated with respect to the baseline control using the comparative cycle threshold method (ΔΔCp).