72°C for 45 s - 1 min. A final extension at 68-72°C for 10 min was performed. Relative expression of specific genes in cells subjected to different treatments was determined by semi-quantitative PCR. The optimal number of cycles required for achieving a linear amplification of serially diluted template was determined, which was then used with other samples to quantify the expression of specific genes. The PCR products were resolved on 1-2% agarose gel containing ethidium bromide and visualized under ultraviolet illumination. The specific primers used are shown in Figure 3.1.

Polymerase chain reaction (PCR) was used to amplify specific nucleotide sequences from eDNA derived from human macrophages. The reaction consisted of Gene Forward primer Reverse primer ER-a 51-GTGGGAATGATGAAAGGTGG-31 51-TCCAGAGACTTCAGGGTGCT-31 51-TGAAAAGGAAGGTT AGTGGGAACC-ER-~ 51-TGGTCAGGGACATCATCATGG-31 31 Bcl-2 51-GTGGAGGAGCTCTTCAGGGA-31 51-AGGCACCCAGGGTGATGCCA-3' Mcl-1 51-CGGCAGTCGCTGGAGATTAT-31 51-GTGGTGGTGGTTGGTTA-31 51-TGGAGTGTCCTTTCTGGTCAACAG-Bfl-1 51-AGCTCAAGACTTTGCTCTCCACC-31 31 iN OS 51-GGCCTCGCTCTGGAAAGA-3 I 51-TCCATGCAGACAACCTT-31 51-CTCCTT AATGTCACGCACGATTTC-Actin 51-GTGGGGCGCCCCAGGCACCA-3 I 31 Figure 3.1. The table shows the forward and reverse pnmers designed agamst specific genes used for amplifying products using PCR. an initial denaturation at 94°C for 4 min, followed by 20-30 cycles of denaturation at 94°C for 30 s, annealing at primer specific temperature for 30 s, and extension at 68

Polymerase Chain Reaction