Background: Tumorigenesis is associated with changes in gene expression and involves many pathways. Dysregulated genes include "housekeeping" genes that are often used for normalization for quantitative real-time RT-PCR (qPCR), which may lead to unreliable results. This study assessed eight stages of hepatitis C virus (HCV) induced hepatocellular carcinoma (HCC) to search for appropriate genes for normalization.
Results: Gene expression profiles using microarrays revealed differential expression of most "housekeeping" genes during the course of HCV-HCC, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB), genes frequently used for normalization. QPCR reactions confirmed the regulation of these genes. Using them for normalization had strong effects on the extent of differential expressed genes, leading to misinterpretation of the results.
Conclusion: As shown here in the case of HCV-induced HCC, the most constantly expressed gene is the arginine/serine-rich splicing factor 4 (SFRS4). The utilization of at least two genes for normalization is robust and advantageous, because they can compensate for slight differences of their expression when not co-regulated. The combination of ribosomal protein large 41 (RPL41) and SFRS4 used for normalization led to very similar results as SFRS4 alone and is a very good choice for reference in this disease as shown on four differentially expressed genes.