β-2 microglobulin is unsuitable as an internal reference gene for the analysis of gene expression in human colorectal cancer

Abstract

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 noncancerous 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. IntroductionInternal control genes are necessary for the accurate determination of gene expression using techniques such as quantitative real-time polymerase chain reaction (PCR). High-throughput real-time reverse transcriptase (RT)-PCR, with its outstanding sensitivity and accuracy, has rendered the selection of a housekeeping (HK) gene used as the internal standard for the estimation and comparison of mRNA levels even more important (1-3). Previous studies showed that the expression levels of commonly used HK genes are different in various tissues or between normal and diseased tissues (1,(4)(5)(6)(7)(8)(9).It has been argued that since the expression levels of various HK genes differ depending on the tissue type or experimental conditions, more than one or even several various control genes should be assessed in parallel for certain tissues (1,3,(10)(11)(12). In colorectal cancer (CRC), downregulation of the HK gene β-2 microglobulin (B2M) has been confirmed using real-time 14).In a previous study, we examined the expression of toll-like receptors 2 (TLR2) and 4 (TLR4) in sporadic human CRC tissue (15). TLRs are known to be involved in innate immunity and to play an important role in immune surveillance (16)(17)(18)(19)(20). In this study, we used a commercially available kit f...

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress

“…b2mg is a component of MHC class I molecules, which are present on all nucleated cells and it has been found to be a suitable reference gene in mouse hematopoetic stem cells [ 31 ], and in K562 cells and leucocytes of normal individuals as well as of malignoma patients [ 32 ] and different tissues of Japanese flounder ( Paralichthys olivaceus ) [ 33 ]. However, in some other studies involving human colorectal cancer cells [ 34 ] and human reticulocyte cells [ 19 ] it has been reported not to be a suitable reference gene.…”

A Bioinformatics-Assisted Review on Iron Metabolism and Immune System to Identify Potential Biomarkers of Exercise Stress-Induced Immunosuppression

“…Both plasma and urinary B2M levels may be reliably and cost-effectively measured [194]. Although it has also been used as a housekeeping or reference gene in exercise and nutrition interventions [195,196], researchers should consider that B2M might be unsuitable for some conditions [197,198]. It has been demonstrated that reduction in the intestinal HEPH and CP-ferroxidase activity may impair Fe absorption and Fe release from intracellular stores, respectively, which decreases Fe levels and results in disturbances of Fe delivery in the bone marrow to support erythropoiesis [199].…”