Abstract
Assessing an optimal reference gene as an internal control for target gene normalization is important during quantitative real time polymerase chain reaction (RT-qPCR) of three dimensional (3D) cell culture. Especially, gene profiling of cancer cells under a complex 3D microenvironment in a polymer scaffold provides a deeper understanding of tumor functioning in vivo. Expression of six housekeeping genes (HKG's): Glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2-microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13) during two dimensional (2D) culture, and alginate-carboxymethylcellulose scaffold based 3D culture conditioned up to 21days was analysed for hepatocellular carcinoma (Huh-7) cells. The gene expression studies were performed by determining primer efficiency, melting curve and threshold cycle analysis. Further, RT-qPCR data was validated statistically using geNorm and NormFinder softwares. The study indicated RPL-13, 18S rRNA and B2M to be stable among selected referral HKG candidates. An exploration of a reliable HKG is necessary for normalization of gene expression in RT-qPCR during varying cell culture conditions.
Highlights
Emerging studies indicate hepatocellular carcinoma (HCC), a primary liver cancer, as one of the leading causes of cancer in the world
Expression of six housekeeping genes (HKG’s): glyceraldehyde-3-phosphodehydrogenase (GAPDH), β-actin (ACTB), beta-2microglobulin (B2M), 18S ribosomal RNA (18S rRNA), peptidyl-propyl-isomerase A (PPIA), and ribosomal protein L13 (RPL-13)) during the monolayer culture, and alginatecarboxymethylcellulose scaffold based three-dimensional (3D) cell culture conditioned up to 21 days was analyzed for hepatocellular carcinoma (Huh-7) cell line
The acridine orange stained hepatocellular carcinoma cells cultivated in 2D (Fig. 1a) showed monolayer formation, and 3D culture during different time intervals of 7, 14 and 21 days showed spheroid formation (Fig. 1b-d)
Summary
Emerging studies indicate hepatocellular carcinoma (HCC), a primary liver cancer, as one of the leading causes of cancer in the world. Application of hepatocellular carcinoma cells for disease modeling in vitro is significantly vital in cancer research due to the dysfunctional liver capacity of HCC in vivo, and to reduce an extensive application of the animal model. A common function of a major internal organ like the liver in vivo is to execute the metabolism of macro/micronutrients, synthesise serum proteins like albumin, and eliminate foreign compounds by transforming them into water-soluble excretions. In this regard, dysfunction of the liver that executes over 500 functions by HCC is a major hurdle for other interrelated functions; in vitro liver disease models are majorly being applied for inventing novel therapeutic avenues. A scaffold-based 3D cell culture allows cells in vitro to grow and proliferate in a manner alike in vivo environment [4]
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