title: β-2 microglobulin is unsuitable as an internal reference gene for the analysis of gene expression in human colorectal cancer.
abstract: It is well-known that gene expression levels should be normalized to a carefully selected and appropriately stable internal control gene. However, numerous studies have demonstrated that the expression of housekeeping (HK) genes, typically used as internal control genes varies considerably. A number of studies have shown that β-2 microglobulin (B2M), an HK gene, frequently used as an internal reference gene, is expressed at low levels in colorectal cancer tissue, when assessed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Due to the fact that the expression levels of various HK genes vary depending on the tissue type or experimental conditions, it has been suggested that several control genes should be analyzed in parallel for certain tissues. In the present study, mRNA expression levels of toll-like receptors 2 (TLR2) and 4 (TLR4) in sporadic human colorectal cancerous and non-cancerous tissues were analyzed relative to three HK genes, β-glucuronidase (GUS), β-actin (BA) and B2M, using a commercially available tool. Relative expression levels were quantified using the three genes individually and together, and TLR2 as well as TLR4 expression was compared in cancerous and non-cancerous colorectal tissue specimens. Consistent data were obtained in most cases when GUS and BA were used as internal control genes. When B2M was used as the internal control gene, TLR2 and TLR4 expression was demonstrated to be higher in cancerous compared to non-cancerous colorectal tissues. These results were consistent with previous observations of low-level B2M expression in cancerous colorectal tissue and suggest that B2M may be inappropriate as an internal control gene for gene expression studies of colorectal cancer.
authors.affiliation: Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501;
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nihon-Yanagi
authors.affiliation: Departments of Research and Development, Sakura Medical Center, Toho University, Chiba 285-0841, Japan.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Terai
authors.affiliation: Departments of Research and Development, Sakura Medical Center, Toho University, Chiba 285-0841, Japan.
authors.forename: Takeyoshi
authors.initials: T
authors.lastname: Murano
authors.affiliation: Departments of Research and Development, Sakura Medical Center, Toho University, Chiba 285-0841, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kawai
authors.affiliation: Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga 849-8501;
authors.forename: Shinya
authors.initials: S
authors.lastname: Kimura
authors.affiliation: Surgery, Sakura Medical Center, Toho University, Chiba 285-0841, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Okazumi
keywords: housekeeping gene; internal control; real-time polymerase chain reaction; β-2 microglobulin