Validation of Suitable Housekeeping Genes for the Normalization of mRNA Expression for Studying Tumor Acidosis

Abstract

Similar to other types of cancer, acidification of tumor microenvironment is an important feature of osteosarcoma, and a major source of cellular stress that triggers cancer aggressiveness, drug resistance, and progression. Among the different effects of low extracellular pH on tumor cells, we have recently found that short-term exposure to acidosis strongly affects gene expression. This alteration might also occur for the most commonly used housekeeping genes (HKG), thereby causing erroneous interpretation of RT-qPCR data. On this basis, by using osteosarcoma cells cultured at different pH values, we aimed to identify the ideal HKG to be considered in studies on tumor-associated acidosis. We verified the stability of 15 commonly used HKG through five algorithms (NormFinder, geNorm, BestKeeper, ΔCT, coefficient of variation) and found that no universal HKG is suitable, since at least four HKG are necessary for proper normalization. Furthermore, according to the acceptable range of values, YWHAZ, GAPDH, GUSB, and 18S rRNA were the most stable reference genes at different pH. Our results will be helpful for future investigations focusing on the effect of altered microenvironment on cancer behavior, particularly on the effectiveness of anticancer therapies in acid conditions.

Keywords: RT-qPCR; acidosis; housekeeping gene; tumor microenvironment.

Figure 1

Transcription profiling of the selected HKG. (A) Heat map showing the relative expression of the selected genes by deep-sequencing analysis performed on MG-63, HOS, and Saos-2 cell lines cultured under acid pH (pH 6.5) compared to physiological medium (pH 7.4) for 24 h. Colors on the heat map indicate the log₂ ratios of expression (representing normalized read counts). Green, downregulation; red, upregulation. RelB is the positive control of the analysis [15]. (B) Box-and-whisker plot indicating range of Cycle threshold (C_t) values of the candidate reference genes in the above mentioned osteosarcoma cells lines in acid or physiological conditions (pH 6.5 and 7.4, respectively). Boxes represent lower and upper quartiles of cycle threshold range with the median indicated as the line across the box; the whiskers represent the 10th and 90th percentiles.

Figure 2

Determination of the optimal number of reference for normalization performed by pairwise variation analysis of candidate HKG under acid pH (A), physiological pH (B) and under both acid and physiological pH (C) culture conditions. A variation coefficient (V) below 0.15 indicates the optimal number of genes required for data normalization.

Figure 2

Determination of the optimal number of reference for normalization performed by pairwise variation analysis of candidate HKG under acid pH (A), physiological pH (B) and under both acid and physiological pH (C) culture conditions. A variation coefficient (V) below 0.15 indicates the optimal number of genes required for data normalization.