Real-time quantitative reverse transcription (RT) PCR analysis

Total RNA was extracted from cells using illustra RNA spin kit (GE healthcare, Buckinghamshire, UK). Single-stranded complementary DNA was synthesized from 1 μg of total RNA using oligo (dT)₁₈ primers and Moloney murine leukemia virus reverse transcriptase (MMLVRT). The complementary DNA for MCP-1, CCL5, IFN-β , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using SsoAdvanced Universal SYBR Green Supermix. All values were normalized to GAPDH mRNA levels. PCR was performed using the following primers:

MCP-1-F; 5′-AAACTGAAGCTCGCACTCTCGC-3′,

MCP-1-R; 5′-ATTCTTGGGTTGTTGAGTGAGT-3′,

CCL5-F; 5′-CTACTCGGGAGGCTAAGGCAGGAA-3′,

CCL5-R; 5′-GAGGGGTTGAGACGGCGGAAGC-3′,

IFN-β-F; 5′-ACTGCCTCAAGGAGAGGATG-3′,

IFN-β-R; 5′-AGCCAGGAGGTTCTCAACAA-3′,

GAPDH-F; 5′-GCACCGTCAAGGCTGAGAAC-3′, and

GAPDH-R; 5′-ATGGTGGTGAAGACGCCAGT-3′.