Figure 1.

Selectivity of the ARE mRNA, IL-8, and suppression of the abundant β-actin mRNA. Total RNA samples (1 μg) from THP-1 or from LPS/cycloheximide- (CHX) treated THP-1 cell line were extracted and subjected to reverse transcriptase (RT) using either Moloney murine leukemia virus (MMLV) or SuperScriptII. Forty ng cDNA was used for the ARE-cDNA PCR at conditions of 10 μM dNTP, 1 Taq U enzyme, and 1 μM of primers. One of the 5′ primers, Ca (Table 1), which contains computationally derived consensus that targets the initiation sites of a subset of ARE-mRNAs that includes IL-8 mRNA, was used along with the 3′ ARE primer that targets the 15-mer ARE region. (a) Aliquots (2 μL) of the amplified ARE products were subjected to relatively stringent polymerase chain reaction (PCR) specific to IL-8 and β-actin (50 μM dNTPs and 25 cycles). (b) Specific PCR to either IL-8 or β-actin was performed to view the relative abundance of the ARE mRNA IL-8 versus the housekeeping β-actin mRNA. Total RNA was used from three different cell lines (1, THP-1; 2, Hela; and 3, WISH). (c) An amount of 4 ng, which mimics the amount of cDNA carried over from the original ARE-cDNA tubes to the second specific PCR tubes, was used as a template. This template was used for second specific PCR for IL-8 and β-actin without the use of ARE-cDNA PCR (lane 1) or with the use of ARE-cDNA PCR (control, lane 2). Arrows indicate the predicted size of IL-8 (289 bp) and β-actin (642 bp). MW: 100-bp size marker.