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Figure 1 (A) NOS III mRNA (cNOS = NOS III) quantification by competitive RNA-PCR. (Top) A constant amount of total RNA was mixed with an increasing number of NOS III competitor RNA molecules, reverse transcribed into cDNA and amplified in duplicate samples by PCR (variation between duplicate samples 5%). The PCR products, indicated as angiotensin-converting enzyme target (643 bp) and NOS III competitor (518 bp), were separated by gel electrophoresis, stained with ethidium bromide and visualized by UV irridiation. On the right-hand side, a molecular weight DNA standard (M) was loaded. (Bottom) The band densities of the NOS III target and competitor DNA were evaluated using a laserdensitometer and a computer-based imaging system. The mean values of duplicate samples were plotted as logarithm of the ratio of competitor to gene target PCR products vs. the logarithm of the known number of competitor molecules. At the competition equivalence point (log ratio = 0) the original number of target mRNAs corresponds to the initial number of competitor RNA molecules used. (B) Restriction analysis of PCR NOS II products, showing specific NOS II fragments. (C) Quantification of NOS III (eNOS) and NOS II (iNOS) gene expression by competitive RT-PCR in nonfailing (NF; n = 5) and failing (CHF; n = 24) LV tissues. The number of transcripts (representing mRNA molecules per microgram total RNA) is depicted.