RESEARCH ARTICLE


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



Hossein Mahboudi1, Negin Mohammadizadeh Heidari2, Zahra Irani Rashidabadi2, Ali Houshmand Anbarestani3, Soroush Karimi4, Kaveh Darabi Darestani5, *
1 Department of medical biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Agronomy and Plant breeding, Agricultural Faculty, Zanjan University, Zanjan, Iran
3 Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
4 Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
5 Biology Department, School of advanced sciences regenerative medicine, Tehran Medical Branch Islamic Azad University, Tehran, Iran


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© 2018 Mahboudi et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Biology Department, School of advanced sciences regenerative medicine, Tehran Medical Branch Islamic Azad University, Kaveh Darabi Darestani, Tehran, Iran; Tel: +989191858816; E-mail: kavehdarabi@ut.ac.ir


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.

Keywords: Absolute, Relative, Copy number, qPCR, Quantification, Monoclonal, Daclizumab.