0.99) as a ranking tool in all 224 human liver tissues examined by 2-DE analysis. Of high importance, ACTB and HSP 60 were successfully validated at both protein and mRNA levels in human hepatic tissues by western blot, immunohistochemistry and real-time quantitative PCR. In addition, no significant correlation of these markers with any clinicopathological features of HCC and cirrhosis was found. Gene stability measure of these two markers with other conventionally applied housekeeping genes was assessed by the geNorm algorithm, which ranked ACTB and HSP60 as the most stable genes among this cohort of clinical samples. Conclusion Our findings identified 2 reference markers that exhibited stable expression across human liver tissues with different conditions thus should be regarded as reliable reference moieties for normalisation of gene and protein expression in clinical research employing human hepatic tissues."/>
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Figure 2 | BMC Cancer

Figure 2

From: A protein-based set of reference markers for liver tissues and hepatocellular carcinoma

Figure 2

Stability validation of potential housekeeping markers in protein and gene levels. (A) Western blot demonstrated the expression stability of beta-actin and HSP 60. An equal amount of 25 μg of protein lysate was loaded per lane in order to measure the expression stability of beta-actin and HSP 60 among liver tissues with different conditions. Representative blot images were showed parallel to the histograms with protein intensities analysed in different liver tissues groups. C, Cirrhosis; < 2 cm, small size [less than (<) 2 cm] paired tumour (T) and non-tumour (NT); > 2 cm, large size [greater than (>) 2 cm] paired tumour (T) and non-tumour (NT). Statistical analysis was performed by one-way ANOVA. Data are presented as the mean ± SD. (B) Immunohistochemical staining showed the localizations and the levels of expression of beta-actin and HSP60 in tumourous, T (n = 30); non-tumourous cirrhotic, NT (n = 30); and normal, N (n = 16) liver tissues. Representative photos are shown (magnification: ×400). (C) Real-time qPCR was performed to evaluate the mRNA levels of beta-actin and HSP60 in tumourous, T (n = 20); non-tumourous cirrhotic, NT (n = 20); and normal, N (n = 16) liver tissues. Statistical analysis was performed by one-way ANOVA (p > 0.05). Data are presented as the mean ± SD.

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