Identification and Validation of Housekeeping Genes for Gene Expression Analysis of Cancer Stem Cells

Abstract

The characterization of cancer stem cell (CSC) subpopulation, through the comparison of the gene expression signature in respect to the native cancer cells, is particularly important for the identification of novel and more effective anticancer strategies. However, CSC have peculiar characteristics in terms of adhesion, growth, and metabolism that possibly implies a different modulation of the expression of the most commonly used housekeeping genes (HKG), like b-actin (ACTB). Although it is crucial to identify which are the most stable HKG genes to normalize the data derived from quantitative Real-Time PCR analysis to obtain robust and consistent results, an exhaustive validation of reference genes in CSC is still missing. Here, we isolated CSC spheres from different musculoskeletal sarcomas and carcinomas as a model to investigate on the stability of the mRNA expression of 15 commonly used HKG, in respect to the native cells. The selected genes were analysed for the variation coefficient and compared using the popular algorithms NormFinder and geNorm to evaluate stability ranking. As a result, we found that: 1) Tata Binding Protein (TBP), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), Peptidylprolyl isomerase A (PPIA), and Hydroxymethylbilane synthase (HMBS) are the most stable HKG for the comparison between CSC and native cells; 2) at least four reference genes should be considered for robust results; 3) the use of ACTB should not be recommended, 4) specific HKG should be considered for studies that are focused only on a specific tumor type, like sarcoma or carcinoma. Our results should be taken in consideration for all the studies of gene expression analysis of CSC, and will substantially contribute for future investigations aimed to identify novel anticancer therapy based on CSC targeting.

Cervical cancer stem‐like cells: systematic review and identification of reference genes for gene expression

“…Corroborating this finding, ACTB instability on cancer studies has already been pointed out by others (Goidin et al, 2001;Rubie et al, 2005;Wang et al, 2016b). Despite being a classical reference gene, ACTB was found to be up or downregulated on several human cancers, including liver, melanoma, renal, colorectal, gastric, pancreatic, esophageal, lung, breast, prostate, ovarian cancers, leukemia, lymphoma, and in CSC of musculoskeletal sarcomas and carcinomas (Guo et al, 2013;Lemma et al, 2016). In addition, cytoskeleton proteins, including b-actin, were regulated and related to epithelial-to-mesenchymal transition in cervical cancer (Shagieva et al, 2016;Wang et al, 2016c).…”

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

“…RPLP0 has been claimed a suitable reference for human intestinal epithelial cells (Dydensborg et al 2006). In turn, PPIA has been repeatedly found a suitable normalizer in a number of human studies (Andrusiewicz et al 2016; Ali et al 2015; Lemma et al 2016), also these concerning CRC patients (Sørby et al 2010; Kheirelseid et al 2010), but affected by cell stimulation in others (Kaszubowska et al 2015). IPO8 and GUSB were yet another HKG recommended for CRC studies (Sørby et al 2010; Blanquicett et al 2002) and highly ranked in our in vitro study as well.…”

Tumor-Activated Mesenchymal Stromal Cells Promote Osteosarcoma Stemness and Migratory Potential via IL-6 Secretion

“…GAPDH was used as housekeeping gene to normalize the expression of the genes of interest [25]. The results were expressed as a ratio between gene of interest and GAPDH gene.…”