Validation of Housekeeping Genes for the Use as Endogenous Controls in Real-Time PCR in Multiple Myeloma
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Multiple myeloma (MM) is a cancer of malignant plasma cells in the bone marrow, and leads to the development of one or more clinical manifestations such as bone destruction, anaemia, and renal insufficiency. The analysis of gene expression is necessary to understand the mechanisms leading to the development of MM. HGF is an important cytokine that has mitogenic, motogenic, or morphogenic effects on different cell types as well as in the pathology of various cancers including MM. Stably expressed housekeeping genes (HKG) are commonly used as endogenous controls to normalize real-time PCR data for gene expression analysis. However, recent studies argue that the expression of these genes may vary under certain experimental conditions. Here, the effects of hypoxia, serum stimulation and c-Myc inhibition on the expression of five HKGs (18S rRNA, β-actin, B2M, GAPDH and TBP) were examined in the three myeloma cell lines ANBL-6, IH-1 and INA-6 in order to validate the reference genes for rt-PCR. The expression of HGF was also studied under same conditions. In hypoxia, cells were incubated for 72 hours under hypoxic condition and in serum induction experiment, cells were serum starved for 24 hours and then induced 10% serum for 8h. In c-Myc inhibition experiment, cells were incubated with c-Myc inhibitor for 24 hours. Total RNA was isolated at different time points and reverse transcribed by normalizing to contain equal amounts of RNA. The resulting cDNA was amplified in rt-PCR for gene expression analysis. The concentration of total RNA was slightly changed under hypoxic conditions and c-Myc inhibition while total RNA was continuously increased over time after serum stimulation. No significant change in gene expression was observed for 18S rRNA, B2M and GAPDH of the cell lines in hypoxia conditions. The espression of 18S rRNA and B2M were not or only slightly affected by serum in the cell lines. Moreover, 18S rRNA was relatively stable and robust during c-Myc inhibition as B2M and β-actin expressed constantly in ANBL-6 and IH-1 cells. The expression of GAPDH and TBP showed variable results under different conditions. HGF was expressed higher in hypoxic conditions in ANBL-6 and IH-1, whereas the expression of HGF was increased in serum induced experiment in ANBL-6 cells. The result of the study confirmed that HKGs variations must be precisely determined before any analysis of a gene expression for each situation. Data from the study showed that the expression of HKGs varied in various experimental treatments and 18S rRNA was the most stable HKG and hence superior for normalization in the analysis of gene expression by using rt-PCR. Additionally, B2M may also be used as an internal control for rt-PCR analysis.