Fig. 2.

Quantitation of TNF-α mRNA expression in human LPS-stimulated and Lassa-infected MDM by competitive PCR. A. Gel-based analysis of TNF-α mRNA expression in LPS-MDM infected with Lassa virus. MDM grown in T25 flasks were stimulated with LPS, infected with Lassa virus (MOI = 5) and incubated for 12 and 24 hours. At 12 and 24 hours p. i. RNA was extracted, converted into cDNA and equivalent amounts of cDNA (based on GAPDH) were amplified. Quantitative PCR was performed with biotin-labeled TNF-α primers using different concentrations of cytokine template (1, 1:10, and 1:100 dilutions) and a constant amount (2,000 copies) of ICS (BioSource). 5 ul-aliquots were analyzed on 2% agarose. M, positions of DNA markers, 500 and 250 bp. B. Quantitation of PCR products. 25 ul from each PCR reaction was denatured under alkaline pH and serial dilutions (1:40–1:320) of TNF-α and ICS amplicons were made in hybridization buffer in ICS- or TNF-α-specific oligonucleotide capture wells. After hybridization the captured amplicons were detected by streptavidin conjugates. Numbers of TNF-α mRNA copies were calculated as follows: Total TNF-α Optical Density (OD)/Total ICS OD × 2 × Input copy number of ICS × cDNA dilution.