Brief report
β-Glucuronidase is a suitable internal control gene for mRNA quantitation in pathophysiological and non-pathological livers

https://doi.org/10.1016/j.yexmp.2013.06.005Get rights and content

Highlights

  • GUSB is the most suitable control gene for quantitative mRNA studies on liver.

  • GUSB showed stable expression in pathophysiological and non-pathological livers.

  • Expression of housekeeping genes was not always stable in diseased liver.

  • It is necessary to statistically determine the best control gene for each exam.

Abstract

The level of expression of housekeeping genes is in general considered stable, and a representative gene such as glyceraldehyde-3-phosphate dehydrogenase is commonly used as an internal control for quantitating mRNA. However, expression of housekeeping genes is not always constant under pathological conditions. To determine which genes would be most suitable as internal controls for quantitative gene expression studies in human liver diseases, we quantified 12 representative housekeeping genes in 27 non-cancerous liver tissues (normal, chronic hepatitis C with and without liver cirrhosis). We identified β-glucuronidase as the most suitable gene for studies on liver by rigorous statistical analysis of inter- and intra-group comparisons. We conclude that it is important to determine the most appropriate control gene for the particular condition to be analyzed.

Introduction

Quantitation of mRNA is routinely used to investigate the molecular pathophysiology of clinical tissue samples. Its application has been accelerated by the development of quantitative real-time PCR that allows even a small clinical sample to be evaluated. To achieve accurate results informative for pathophysiology, that is, independent of the quality of the RNA and the efficiency of cDNA synthesis, it is customary to normalize the quantity of target gene mRNA against an internal control gene such as a housekeeping gene. The 18S rRNA (RNA18S5), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin genes are frequently used as an internal control, but some of the housekeeping genes are not constantly expressed in different tissues with and without disease. For example, Rubie et al. (2005) reported that GAPDH was up-regulated in cancerous tissues of pancreas, stomach, colon and colorectal liver metastases compared with normal tissues. Boujedidi et al. (2012) examined livers from patients with alcoholic liver disease and demonstrated that GAPDH mRNA decreased in the steatotic liver whereas it increased in the cirrhotic liver. Thus, it would appear necessary to individually select a control gene suitable for mRNA quantitation, which would differ in different experiments. Here, we focused on liver disease, and determined which gene was most appropriate as a control for non-cancerous liver at different stages of hepatitis.

Liver diseases are mainly caused by infection with viruses such as hepatitis B virus and hepatitis C virus (HCV). Persistent infection with these viruses results in the development of chronic hepatitis, liver cirrhosis and finally hepatocellular carcinoma. The host defense response against viral infection is involved in viral proliferation and viral pathogenesis. Secondary responses, such as inflammation, are closely related to the severity of disease. The viral infection and subsequent development of liver disease influence the expression of various genes including even the housekeeping genes in the liver. Studying alterations of mRNA expression and their linkage networks is useful for investigating mechanisms of disease development due to viral infection. To do so, it is necessary to determine which gene to use as an internal control for the normalization of mRNA quantitation in livers with different diseases. Although there are a few reports investigating the expression of housekeeping genes in HCV-positive liver disease, the results have not been consistent (Congiu et al., 2011, Romanowski et al., 2008, Waxman and Wurmbach, 2007). In particular, there are few studies comparing normal liver and chronic hepatitis C livers. In the present study, to determine an adequate internal control gene for mRNA quantification to compare normal and chronic hepatitis C liver with and without cirrhosis, we investigated which of the 12 selected housekeeping genes was most constantly expressed and therefore most appropriate as an internal control.

Section snippets

Specimens

Non-cancerous liver tissues were obtained from 32 cases of hepatocellular carcinoma positive for hepatitis C virus by surgical resection. The tissues were histologically examined and the degree of fibrosis was classified according to the New Inuyama Classification, as follows: F0, no fibrosis; F1, fibrous portal expansion; F2, bridging fibrosis; F3, bridging fibrosis with lobular destruction; F4, liver cirrhosis (Ichida et al., 1996). Based on the fibrosis score, the specimens were classified

Standard deviation of expression of 12 housekeeping genes

Pathophysiological stratification of the livers in this study assigned to each one of the three groups: chronic hepatitis 1) with and 2) without liver cirrhosis caused by HCV infection and 3) normal liver. These groups were designated LC, CH and NL, respectively. The mRNAs of 12 selected housekeeping genes were quantified, and normalized against the median of all tested samples. The quantity of each mRNA was compared among the three groups and with the total group, ALL (Fig. 1). To determine

Discussion

We have identified β-glucuronidase as the most suitable gene for studies on liver by rigorous statistical analysis of inter- and intra-group comparisons. Meanwhile, we have also identified 6 housekeeping genes which should not be used as internal controls, based on their significantly different expression in normal and disease groups. These are GAPDH, SMARCA5, TBP, B2M, GMFG and RNA18S5, of which the latter is commonly used as a reference gene. However, it was up-regulated in the CH group in

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the Nihon University Joint Research Grant for 2006, Grant-in-Aid for Scientific Research (C) 22590350 from MEXT (2010), “Strategic Research Base Development” Program for Private Universities subsidized by MEXT (2010), and by a donation from the Nippon Flour Mills Company, Ltd. We are indebted to Prof Norio Kokudo, Graduate School of Medicine, University of Tokyo and Dr Takuichi Oikawa, Nihon University School of Medicine for providing data on the patients and to Prof

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