Reconsidering Real-Time PCR Method for Gene Expression Evaluation in Cancer Based on Cell-Type Normalization: A Methodological Perspective

Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely used technique for assessing gene expression in both experimental and clinical specimens. One of its major applications is the investigation of gene expression profiles in cancer, where the expression levels of target genes are compared under specific biological conditions, such as between tumor and non-tumor tissues. To ensure accurate and reliable comparisons, reference genes, commonly referred to as housekeeping genes, are used to normalize variations. However, an important question remains: Is normalization using housekeeping genes alone sufficient to ensure the accuracy and reliability of gene expression analyses in cancer? Recently, methods like single cell RNA sequencing have shown that there is a heterogeneity of tumor and non-tumor cells in tumor samples. In fact, even in a single tumor specimen, there is a heterogenous of normal cells such as macrophages, lymphocytes, and neural cells, beside the heterogeneity of tumor cells. This intratumoral cellular complexity may influence the interpretation of gene expression data obtained using RT-qPCR. Accordingly, I propose a new normalization strategy called “cell-type normalization” according to the different types of cells in RT-qPCR besides cell number normalization. This approach could potentially be implemented using the cell-type specific markers. Testing this hypothesis requires identification of suitable genes in each sample type followed by systematic experimental assessment of the method's accuracy, reproducibility, and applicability.