Computational protocol: Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses

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Protocol publication

[…] Statistical analyses were performed with SPSS 15.0 for Windows (SPSS Software, Chicago, IL, USA). P values <0.05 were considered statistically significant. For stability comparison of candidate reference genes, we applied the software geNorm, version 3.4 [], and NormFinder []. The program geNorm is a Visual Basic application tool for Microsoft Excel and is available on the Internet upon request by the programmers. CT values were converted into relative quantities for analysis with geNorm []. The program selects from a panel of candidate reference genes the two most stable genes or a combination of multiple stable genes for normalization. The NormFinder is also freely available on the Internet It is a Microsoft Excel add-in and calculates the stability values of the individual candidate reference genes for normalization []. The stability value is based on the combined estimate of intra- and inter-group expression variations of the genes studied. A low stability value indicating a low combined intra- and inter-group variation proves high expression stability. Using this approach, the most stable single gene is calculated and an additional combination of two genes is recommended because the stability value of that combination is generally lower than that of the single gene. […]

Pipeline specifications

Software tools geNorm, NormFinder
Application qPCR
Organisms Hepatitis B virus, Homo sapiens
Diseases Hepatitis B, Carcinoma, Hepatocellular, Neoplasms