Hybridization buffer systems impact the quality of filter array data

doi:10.1016/j.vascn.2004.02.001

Journal of Pharmacological and Toxicological Methods

Volume 50, Issue 1, July–August 2004, Pages 67-71

https://doi.org/10.1016/j.vascn.2004.02.001 Get rights and content

Abstract

Introduction: cDNA microarray technology has greatly facilitated mechanistic studies in pharmacology and toxicology. A clean hybridization with minimal background is critical for successful microarray analysis and is highly desired. However, clean hybridization alone is not enough; verification is needed. Methods: Total RNA was isolated from the livers of acetaminophen-intoxicated mice and was subjected to cDNA microarray analyses using ExpressHyb, ULTRArrayHyb or MicroHyb on nylon membranes. Real-time RT-PCR analyses were performed for verification. Results: We have demonstrated in this paper that hybridization systems can significantly impact the quality of array data. MicroHyb produced very clean hybridizations, but some results could not be confirmed by real-time RT-PCR and in accord with biological responses. The hybridization images from ExpressHyb were not always clean, but were reliable. The sensitivity of ULTRArrayHyb was moderate. Conclusion: This study has indicated the importance of selecting hybridization buffers in membrane arrays and recommended real-time RT-PCR for follow-up analysis. Gene expression changes should also be correlated with biological significance.

Introduction

The cDNA microarray has been a huge technical advance in biological research because it allows the simultaneous measurement of hundreds to thousands of genes. Two main types of microarray, the chip-array (glass) and filter-array (nylon membrane), are currently used for gene expression analysis. A clean hybridization with minimal background is critical for successful microarray analyses, and the need for the optimization of hybridization systems is a major part of experimental protocols, which leads to the development of various hybridization systems. Microarray hybridizations can be influenced by many factors. Here, we compared the three commercially available hybridization systems in membrane arrays and found that different hybridization systems could make a substantial difference in the quality of data from membrane arrays, using gene expression analysis of acetaminophen hepatotoxicity as an example.

Section snippets

Animals and treatment

Adult male CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA) and were housed in facilities accredited by the American Association for the Accreditation of Laboratory Animal Care at the National Institute of Environmental Health Sciences, at 20–22 °C with a 12-h light/dark cycle. The animals were given free access to rodent chow and tap water. The mice were given a hepatotoxic dose of acetaminophen (APAP, 600 mg/kg ip for 8 h), and the livers were removed for array

Microarray images

The representative hybridization images are shown in Fig. 1. Note the following: (1) The images were clean, but the patterns of the gene expression are quite different between the three hybridization systems. (2) The acetaminophen-induced expressions of genes encoding the acute phase proteins, such as c-Jun, heme oxygenase-1 (HO-1), early response growth factor-1 (ERG1), heat shock protein 70 (HSP70), the increased expression of DNA damage protein GADD45, and the decreased expression of CYP2E1,

Discussion

This study provides a lesson we have learned from our own mistakes. In an attempt to get a clear hybridization image, we tested five different hybridization systems and found that MicroHyb system was the best and always gave very clean hybridization images, while the ExpressHyb did not always produce clean images. We have thus switched to MicroHyb system for over 150 arrays in five different projects. The hybridizations of every membrane with MicroHyb were very clean and highly reproducible.

Acknowledgements

The authors thank Drs. Kartik Shankar, Tong Zhou, Yaxiong Xie, and Alex Merrick for their valuable help and comments for this technical appraisal.

References (5)

J. Liu et al.
The nitric oxide donor, V-PYRRO/NO, protects against acetaminophen-induced hepatotoxicity in mice
Hepatology
(2003)
J. Liu et al.
Toxicogenomic analysis of aberrant gene expression in liver tumors and non-tumorous livers of adult mice exposed in utero to inorganic arsenic
Toxicological Sciences
(2004)

There are more references available in the full text version of this article.

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Citation Excerpt :
The relative differences in gene expression between groups were expressed using cycle time (Ct) values; these Ct values of the interested genes were first normalized with that of GAPDH in the same sample, and then the relative differences between control and treatment groups were calculated and expressed as relative increases setting the control as 100%. Assuming that the Ct value is reflective of the initial starting copy and that there is 100% efficiency, a difference of one cycle is equivalent to a two-fold difference in starting copy (Liu et al., 2004a,b; Livak and Schmittgen, 2001; Walker, 2001). All data are expressed as mean ± SE.
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Brief communicationHybridization buffer systems impact the quality of filter array data

Abstract

Introduction

Section snippets

Animals and treatment

Microarray images

Discussion

Acknowledgements

Hepatology

Toxicogenomic analysis of aberrant gene expression in liver tumors and non-tumorous livers of adult mice exposed in utero to inorganic arsenic

Toxicological Sciences

Brief communication
Hybridization buffer systems impact the quality of filter array data