Quantitation of DNA from flow cytometry analysis using fluorescent probe

Akon Higuchi, Yukiyo Murayama, Yasuhisa Tsukamoto, Toshinori Kojima, Hanako Kitamura

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

A reproducible method to quantify DNA in DNA and protein solutions at ppb levels using flow cytometry analysis is reported herein. After DNA and protein solutions were dyed with fluorescent probe (PicoGreen™) intercalating, flow cytometric scattergrams of DNA at fluorescent intensities of 525 nm and 575 nm showed a higher intensity of emission at 525 nm than those of proteins. DNA quantitation was performed using calf thymus DNA with two standard curves. The high standard curve showed a semi-logarithmic plot from 1 to 50 ppb with a correlation coefficient of 0.993, whereas the low standard curve described the second-power equation from 0.1 to 5 ppb DNA with a correlation coefficient of 0.998. A flow cytometry analysis of the DNA concentration was powerful for estimating the DNA concentration in a protein solution containing salts, such as saline, because the present flow cytometry analysis showed less than a 37% decline when the DNA solution contained 0.15 mol/L NaCl. The DNA content in albumin, cellulase, γ-globulin, and pepsin was estimated to be 0.078 ± 0.006, 0.26 ± 0.04, 3.0 ± 0.4, and 1.2 ± 0.1 μ-DNA/g-protein (n = 4), respectively, based on flow cytometry analysis of the protein solutions.

Original languageEnglish
Pages (from-to)2021-2025
Number of pages5
JournalBulletin of the Chemical Society of Japan
Volume77
Issue number11
DOIs
StatePublished - 2004

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