Commentary on a Classic JHC Article on the Histochemical Measurement of DNA Content in Cells

Abstract

This article comments on the significance of a highly cited review article on DNA cytochemical quantitation that was published in the Journal of Histochemistry and Cytochemistry in 2002 (David C. Hardie, T. Ryan Gregory, and Paul D.N. Hebert. From pixels to picograms: A beginners’ guide to genome quantification by Feulgen image analysis densitometry.

The accompanying review article by Hardie et al. ¹ on Feulgen DNA staining and quantification is a Journal of Histochemistry & Cytochemistry classic in the true sense of the word. The review was published in 2002 and has been cited over 200 times since. Moreover, it is a classical cytochemistry article describing the usefulness of one of the oldest staining reactions that was introduced by Robert Feulgen in 1924. This article is an example of thorough methodological investigations to standardize and validate the staining method for quantitative cytochemical analysis of the total amount of DNA in individual cells using image analysis. It has been written in a clear style and discusses all potential pitfalls and sources of errors with respect to quantitative cytochemistry such as background absorbance, glare, compactness of the DNA in nuclei, cell proliferation, and so on.

The optimal staining and quantification procedures are presented in a step-by-step fashion in appendices at the end of the article. Table and figures are included to demonstrate the quantitative properties of the Feulgen reaction to stain DNA in individual cell nuclei and the effects of potential pitfalls. Therefore, the article has not lost its impact in the past 20 years.

The authors did an excellent and timely job to meet the aim of the review as formulated by the authors themselves:

. . . this is the review we wish we had had when we began our own animal genome size measurements . . . to permit broad comparative analyses of genome size diversification among taxa and thereby illuminate the mechanisms and time scales involved. Only when this is accomplished will a truly comprehensive analysis of eukaryotic genome evolution be possible.

The authors hit the nail on its head, and this warrants the highlighting and reprinting of their review article. It also demonstrates to all scientists the necessity of using standardized and validated histochemical or cytochemical methods particularly when they cannot perform the standardization and validation themselves.