Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

<p>Abstract</p> <p>Background</p> <p>Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.</p> <p>Methods</p> <p>Six genes, beta-2-microglobulin (<it>B2M</it>), glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), hydroxymethyl-bilane synthase (<it>HMBS</it>), hypoxanthine phosphoribosyl-transferase 1 (<it>HPRT1</it>), succinate dehydrogenase complex, subunit A (<it>SDHA</it>) and ubiquitin C (<it>UBC</it>), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both <it>geNorm </it>and <it>NormFinder </it>software.</p> <p>Results</p> <p><it>HMBS </it>and <it>GAPDH </it>were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. <it>HMBS, GAPDH </it>and <it>UBC </it>were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of <it>HMBS, B2M</it>, <it>SDHA </it>and <it>GAPDH </it>was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.</p> <p>Conclusion</p> <p>Of six genes studied, <it>HMBS </it>was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.</p

Long-Term Renal Function in Liver Transplant Recipients After Conversion From Calcineurin Inhibitors to mTOR Inhibitors

BACKGROUND: Renal dysfunction often occurs in liver transplant (LT) recipients receiving calcineurin inhibitor (CNI)-based immunosuppressive regimens, increasing morbidity and mortality rates. Replacement of CNIs by mTOR inhibitor-based immunosuppressive protocols may prevent renal impairment in LT recipients. MATERIAL AND METHODS: Outcomes in patients who underwent LT between 1996 and 2010 at our center and who were switched from CNI-based to mTOR inhibitor-based immunosuppression were retrospectively analyzed. Renal course, hyperlipidemia, and graft rejection were assessed in patients maintained on this CNI-free regimen for at least 24 months. RESULTS: Of the 85 patients switched from CNI-based to mTOR inhibitor-based, CNI-free immunosuppression, 78 met the inclusion criteria. Within the first 6 weeks after switching, the covariable adjusted estimated glomerular filtration rate (eGFR) increased 5.6 mL/min [95% confidence interval 2.6–8.7 mL/min, p<0.001], but there were no further statistically noticeable changes in eGFR. Concentrations of cholesterol and triglycerides increased statistically, noticeable within the first 12 months after drug conversion. Histologically proven graft rejection was observed in 4 patients (5.1%) after conversion. CONCLUSIONS: Conversion from CNI-based to CNI-free, mTOR inhibitor-based immunosuppression after LT is safe and can result in significant renal recovery. CNI-free, mTOR inhibitor-based immunosuppression is a potential option for patients with contraindications for CNIs and for LT recipients with rapid reduction in kidney function due to CNIs

BMC Cancer BioMed Central

Research article Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PC