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Real Time RT-PCR

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

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Published on: February 25, 2017

Evaluation of Plasma-Derived Cell-Free RNA isolation methods using PCR-Based quantification.

Gizem Inetas-Yengin¹, Zehra Omeroglu-Ulu¹, Fikrettin Sahin¹

¹Department of Genetics and Bioengineering, Yeditepe University, Inonu Mah. Kayisdagi Cad, Atasehir, Istanbul, 34755, Turkey.

Molecular Biology Reports

|January 3, 2026

Summary

Commercial kits offer superior cell-free RNA (cfRNA) isolation from plasma compared to traditional methods. Optimized isolation and reference gene selection are key for reliable RNA biomarker discovery.

Keywords:

Cell-free RNA Digital PCR Liquid biopsy Plasma RNA derivatives

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Area of Science:

Biochemistry
Molecular Biology
Genomics

Background:

Cell-free nucleic acids (cfNAs) in body fluids are promising biomarkers.
cfRNAs from blood offer minimally invasive disease diagnosis.
cfRNA isolation faces challenges like degradation and contamination.

Purpose of the Study:

To compare the purity and quality of cfRNAs isolated from plasma.
To evaluate five commercial cfRNA isolation kits against traditional methods.
To assess the reliability of cfRNA isolation for biomarker discovery.

Main Methods:

Evaluated five commercial cfRNA isolation kits using plasma.
Assessed RNA purity and quality via digital PCR (dPCR) and quantitative PCR (qPCR).
Measured cfRNA expression levels using reference genes (GAPDH, B2M).

Main Results:

Commercial kits demonstrated superior cfRNA isolation compared to phenol-chloroform methods.
GAPDH showed greater consistency as a reference gene than B2M.
Both GAPDH and B2M reference genes exhibited statistically significant expression.

Conclusions:

Commercial cfRNA isolation kits are more effective than phenol-chloroform procedures.
Optimized isolation strategies are crucial for reliable cfRNA biomarker discovery.
Careful selection of reference genes enhances the accuracy of cfRNA analysis.