Prospect and Competence of Quantitative Methods via Real-time PCR in a Comparative Manner: An Experimental Review of Current Methods

Abstract

Background: There are numerous approaches dealing with relative and absolute quantitation. The methods differ in their efficiency assumption and applicability. Objective: Current methodologies and rations used in qPCR quantification were compared in an experimental study of transgenic copy number determination of a monoclonal antibody Daclizumab. Methods: With an inter and intra-methodical view, variations in relative and absolute quantification strategies were discretely extracted and compared to one another. Results: In relative quantification, six methods were studied and the ratios were computed relative to Glucagon as internal control. For Absolute quantification, the calculations were based on standard curve. Relative quantification considers the relative changes in expression levels while Absolute quantification relates the PCR signal to input copy number with a calibration curve. Conclusion: The observed unevenness of the ratios in Relative approach pointed mainly to the efficiency changes and its calculation formula. Whereas results in Absolute approach strategies showed homogeneity which indicates the consistency of the calculation method.

An Association between Insulin Resistance and Neurodegeneration in Zebrafish Larval Model (Danio rerio)

“…Amplified signals from duplicate reactions were detected and analyzed using the MiniOpticon BioRad analyzer (California, USA) and CFX manager software (California, USA). The amplification protocol used was as follows: initially, withhold phase at 95 °C for 5 min denaturation and enzyme activation, perform 40 cycles of 95 °C for 3 s and 61 °C for 30 s, and set the melting curve phase to 65 °C for 60 s and 95 °C for 15 s. The housekeeping gene for all primers was beta-actin, with qPCR data normalized to the beta-actin levels using the Livak method with the ΔΔCt value [ 75 ]. Three independent experiments were performed, and the quantitative data obtained were averaged based on the quantification cycle (Cq) values, which were used to calculate the relative expression gene.…”

Effect of Ramipril on Endothelin-1 Expression in Myocardial Tissue at Wistar Rats Induced Myocardial Infarction

“…ACE-I was able to reduce the formation of the potent vasoconstrictor ET-1 and increase NO bioavailability in HUVECs. In turn NO represents a barrier against oxidants such as unscavenged superoxide anion, decrease generation of reactive oxygen species induced by TNFα in HUVECs and decrease LDL susceptibility to oxidation [9], [10], [11]. ACE-I completely prevented NO-deficient that responsible for vasodilative, antiagregative, and antiproliferative action to prevent the acute myocardial infarction [12].…”