De-regulation of common housekeeping genes in hepatocellular carcinoma.
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.


CONCLUSIONS
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.
Waxman
Samuel
S
Mount Sinai School of Medicine, Department of Medicine, Division of Hematology/Oncology, New York, NY, USA. Samuel.Waxman@mssm.edu
Wurmbach
Elisa
E
D000199:Actins / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000821:virology; D002472:Cell Transformation, Viral; D015536:Down-Regulation; D016326:Extracellular Matrix Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D020890:Neuregulin-1; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D016601:RNA-Binding Proteins / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D000068103:Serine-Arginine Splicing Factors; D035181:TATA-Box Binding Protein / Q000235:genetics; D015854:Up-Regulation
D000199:Actins; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D008565:Membrane Proteins; C094131:NRG1 protein, human; D009419:Nerve Tissue Proteins; D020890:Neuregulin-1; C446290:PRIMA1 protein, human; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C081934:SRSF4 protein, human; D035181:TATA-Box Binding Protein; C098946:hyaluronan-mediated motility receptor; C029611:ribosomal protein S20; C076844:RPL41 protein, human; D000068103:Serine-Arginine Splicing Factors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C507768:IRAK1 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases