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.
Highlights
One of the demanding methods to quantify gene expression is Real-time Polymerase Chain Reaction [1 - 5]
The logic behind qPCR is detecting the amount of the amplified genomic product in real time by means of various chemicals (e.g.specific DNA binding dyes, hydrolysis probe, molecular beacon etc.), which leads to creation of a florescent sigmoid curve [10]
Relative quantification designates the variation in expression of the target gene relative to some reference group [10, 13 - 16]
Summary
One of the demanding methods to quantify gene expression is Real-time Polymerase Chain Reaction [1 - 5]. The technique quantifies the target genomic region and at the same time holds the detection potential. The logic behind qPCR is detecting the amount of the amplified genomic product in real time by means of various chemicals (e.g.specific DNA binding dyes, hydrolysis probe, molecular beacon etc.), which leads to creation of a florescent sigmoid curve [10]. There are main steps in determining copy number via qPCR, including: choosing the appropriate genomic region, choosing specific primers, practical validation of the primers, making accurate serial dilution of genomic DNA, and selecting an appropriate detecting chemicals [11]. Relative quantification designates the variation in expression of the target gene relative to some reference group [10, 13 - 16]. It means showing fold changes in expression between two samples.
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