Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification

Area of Science:

• Biochemistry
• Molecular Biology
• Clinical Diagnostics

Background:

• Quantifying circulating free DNA (cfDNA) in patient serum is crucial but challenging.
• Quantitative real-time PCR (QPCR) is effective but costly and requires specialized equipment.
• Exploring simpler, cost-effective methods for cfDNA quantification is essential for routine use.

Purpose of the Study:

• To evaluate the efficiency of direct fluorescent dye-based DNA quantification (PicoGreen) for serum cfDNA.
• To compare PicoGreen quantification with established QPCR methods using specific gene targets.
• To determine if PicoGreen is a viable, less expensive alternative for routine cfDNA analysis.

Main Methods:

• Serum samples from 180 cancer patients and 58 healthy volunteers were analyzed.
• DNA quantification was performed using three methods: QPCR targeting beta-globin, QPCR targeting cyclophilin A, and PicoGreen DNA quantification kit.
• QPCR methods used specific standard curves for accurate copy number determination.

Main Results:

• Healthy controls had <160 cyclophilin gene copies/mL; cancer patients ranged from 300 to 215,000 copies/mL.
• The cyclophilin A QPCR method strongly correlated with both beta-globin QPCR (r=0.911) and PicoGreen quantification (r=0.915).
• PicoGreen DNA quantification showed high correlation with QPCR-based methods.

Conclusions:

• Direct PicoGreen quantification is a less time-consuming and inexpensive method for serum cfDNA analysis.
• PicoGreen quantification is well-correlated with QPCR, offering a practical alternative for routine clinical laboratories.
• This method overcomes the limitations of QPCR, such as cost and equipment requirements.