Influence of reagent formulation on mRNA quantification by RT-PCR using imported external standard curves.

Use of an imported external standard curve is common in real-time quantitative RT-PCR. Two practical strategies for long-term experiments include importing a grand mean standard curve to all accumulated runs or using daily imported standard curves, fixing the slope at the beginning of the experiment and calibrating successive runs with curves generated from this imported slope, adding a single standard that registers the variation in the y-intercept. This study determines the influence that a change in reagent lots has on these two calibration approaches when determining mRNA copy numbers of the ornithine decarboxylase and porphobilinogen deaminase genes. Two sets of determinations were run with the use of lot A and lot B. A marked decrease in the crossing points (Cp) in the standards for both genes at all concentration levels was observed with the change in lots. A grand mean standard curve was generated for each gene and each set and comparisons between the sets were performed. Statistically significant differences were found with respect to the y-intercept but not the slope, suggesting that the change of reagent lot affected the detection sensitivity but not the efficiency of the reaction. The excellent correlation coefficients obtained for these curves for each gene were not achieved when overall data from both sets were combined to generate an overall grand mean standard curve. We conclude that when faced with a change of RT-PCR reagent lot that will affect the detection sensitivity of the method, samples should be calculated with either the daily imported standard curves or with the respective grand mean standard curve for each lot.

Antioxidant effect of polyamines on erythrocyte cell membrane lipoperoxidation after free-radical damage.

This in vitro study determined the effect of three doses each (100, 500 and 1000 microm) of putrescine, spermidine and spermine on malondialdehyde (MDA) release in red blood cells (RBCs) from healthy individuals after hydrogen peroxide stimulation (10 mM). Twenty-two volunteers, 9 males and 13 females, aged 41.5 +/- 16.4 years, were studied. MDA was measured by thiobarbituric reaction (TBARs) and the results were calculated using epsilon = 1.56 x 10(5). The analysis of variance (ANOVA) demonstrated a statistically significant overall decrease in MDA release in the polyamine-exposed cells (p < 0.0001) when compared with unexposed cells. Individual analysis of each polyamine separately showed a 52% decrease in MDA release with added spermine and a 39.5% decrease with added spermidine (p < 0.001). No evaluable effect was found for putrescine. There was no correlation between the effect produced and the three doses of spermidine or spermine added, indicating a non dose-dependent action.