Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold
Background 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. Methods and Results 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 21 days 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. Conclusion An exploration of a reliable HKG is necessary for normalization of gene expression in RT-qPCR during varying cell culture conditions..
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:49 |
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| Enthalten in: |
Molecular biology reports - 49(2021), 1 vom: 19. Okt., Seite 797-804 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Badekila, Anjana Kaveri [VerfasserIn] |
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| Links: |
Volltext [lizenzpflichtig] |
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| Themen: |
3D culture |
| Anmerkungen: |
© The Author(s), under exclusive licence to Springer Nature B.V. 2021 |
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| doi: |
10.1007/s11033-021-06830-y |
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OLC2077772328 |
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| 520 | |a Background 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. Methods and Results 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 21 days 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. Conclusion An exploration of a reliable HKG is necessary for normalization of gene expression in RT-qPCR during varying cell culture conditions. | ||
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