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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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Updated: Jun 25, 2025

NanoDrop Microvolume Quantitation of Nucleic Acids

NanoDrop Microvolume Quantitation of Nucleic Acids

Published on: November 22, 2010

PicoGreen assay for nucleic acid quantification - Applications, challenges, and solutions.

Eunmi Ban¹, Aeri Kim¹

¹College of Pharmacy, CHA University, Seongnam, 13488, South Korea.

Analytical Biochemistry

|May 24, 2024

Summary

PicoGreen offers a rapid, cost-effective method for nucleic acid quantification, overcoming limitations of traditional techniques. This review highlights its applications and strategies to address experimental challenges for accurate DNA and RNA analysis.

Keywords:

Analytical method Fluorescence intensity Nucleic acids PicoGreen

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Area of Science:

Molecular Biology
Biochemistry
Analytical Chemistry

Background:

Traditional nucleic acid analysis methods like gel electrophoresis and qRT-PCR are time-consuming and costly.
Limitations exist for small RNA detection and low concentrations with standard techniques.
PicoGreen (PG) presents an alternative fluorescent dye for nucleic acid quantitation.

Purpose of the Study:

To review recent applications of PicoGreen for nucleic acid quantification.
To evaluate PG as a rapid, cost-effective, robust, and accurate assay.
To discuss PG's limitations and potential solutions for broader application.

Main Methods:

PicoGreen (PG) fluorescence assay for double-stranded DNA (dsDNA) and RNA quantitation.
Application in solution and within cells using FACS and fluorescence microscopy.
Exploration of experimental factors influencing PG fluorescence intensity.

Main Results:

PG enables accurate quantification of dsDNA and dsRNA, including small RNAs like miRNA mimic and siRNA.
PG is effective for both in vitro and in-cell nucleic acid analysis.
Fluorescence intensity is sensitive to pH, salts, and chemical reagents, requiring optimization.

Conclusions:

PicoGreen is a versatile tool for rapid and cost-effective nucleic acid quantification.
Understanding and mitigating PG's sensitivity to experimental conditions is key to its robust application.
Further research can expand PG's utility in diverse biological and formulation analyses.