INOS cDNA

Figure 5. Effect of HMP on mRNA expression of iNOS and COX-2. Total RNA isolatedfrom RA W 264.7 cells after 12 hrs of treatments with IPS either alone or with various concentration of HMP, was reverse transcribed to make cDNA and 2 pi (for iNOS) or 1 pi (for COX-2) of cDNA was amplified using gene specific primer (Ambion Inc). Equal volume (10 pi) of each PCR reaction was resolved in 2 % agarose gel Lane /, control 2. LPS (0.5 pg/ml) 3, LPS+HMP (12.5 pM) and Lane, 4 LPS HMP (25 pM).

Cells were washed with ice-cold PBS and total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction (27). 5 pg total RNA was reverse-transcribed (RT) into cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase and oligo (dT)i primer by incubating the reaction mixture (15 pL) at 37 C for 90 min. The polymerase chain reaction (PCR) was performed as described previously (22). A final volume of 25 pL contained dNTPs (each at 200 pM), IX reaction buffer, 0.4 pM each primer (iNOS forward ... 

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