Abstract
Objectives. Breast cancer (BC) cell lines MCF-7 representing luminal A subtype and characterized by PR+, ER+ & HER2-and MDA-MB-231 representing triple negative BC subtype, are two of the most frequently researched and published cell lines in breast cancer research. Advanced molecular techniques like quantitative real time PCR (RT-qPCR) are routinely employed to study gene expression in the cell lines. However, to overcome sampling errors, the results from qPCR should be normalized using a set of reference genes. Reference genes (previously called housekeeping genes) are endogenous genes that are thought to have stable expression across cell lines and tissues. Nonetheless, evidence from literature points otherwise. Therefore, identification of reference genes is important to avoid incorrect normalization of qPCR data and preclude fallacious conclusions.
Materials and Methods Two parallel cultures of MCF-7 and two of MDA-MB-231 cell lines were cultured in DMEM/F12 with 10% FBS. Three lysates per passage were collected over multiple passages from each culture and RT-qPCR was performed. Seven endogenous reference genes were used for qPCR: ACTB, GAPDH, PGK1, RPL13a, 18SRNA, 28SRNA and HSPCB. Gene expression was analyzed using NormFinder, geNorm and RefFinder.
Results NormFinder suggested GAPDH and ACTB as the most stable gene pair in MCF-7 while HSPCB and RPL13a were reported to be the most stable gene pair in MDA-MB-231. geNorm indicated in both the cell lines that addition of a third reference gene for normalization would not affect the results as the V2/3 was below the recommended cutoff of 0.15 (V2/3 = 0.0023 and 0.0009 respectively). RefFinder also indicated similar results.
Conclusions Differences in the endogenous gene expression across the BC cell lines and passages during long term cultivation represents the need for identifying the suitable reference genes as a prerequisite in experiments relating to gene expression levels to preclude incorrect normalization.
Materials and Methods Two parallel cultures of MCF-7 and two of MDA-MB-231 cell lines were cultured in DMEM/F12 with 10% FBS. Three lysates per passage were collected over multiple passages from each culture and RT-qPCR was performed. Seven endogenous reference genes were used for qPCR: ACTB, GAPDH, PGK1, RPL13a, 18SRNA, 28SRNA and HSPCB. Gene expression was analyzed using NormFinder, geNorm and RefFinder.
Results NormFinder suggested GAPDH and ACTB as the most stable gene pair in MCF-7 while HSPCB and RPL13a were reported to be the most stable gene pair in MDA-MB-231. geNorm indicated in both the cell lines that addition of a third reference gene for normalization would not affect the results as the V2/3 was below the recommended cutoff of 0.15 (V2/3 = 0.0023 and 0.0009 respectively). RefFinder also indicated similar results.
Conclusions Differences in the endogenous gene expression across the BC cell lines and passages during long term cultivation represents the need for identifying the suitable reference genes as a prerequisite in experiments relating to gene expression levels to preclude incorrect normalization.
| Original language | English |
|---|---|
| Pages | 42-42 |
| Number of pages | 1 |
| Publication status | Published - 27 Mar 2020 |
| Event | 6th RSU International Student Conference 2020: Health and Social Sciences - Rīga Stradiņš University, Rīga, Latvia Duration: 27 Mar 2020 → 28 Mar 2020 Conference number: 6 https://drive.google.com/file/d/1T4P2qTeS9Q-FpWavIifEb_v6Hu9RI8rH/view https://isc.rsu.lv/about/past-conferences/ |
Conference
| Conference | 6th RSU International Student Conference 2020 |
|---|---|
| Abbreviated title | ISC 2020 |
| Country/Territory | Latvia |
| City | Rīga |
| Period | 27/03/20 → 28/03/20 |
| Internet address |
Field of Science*
- 3.1 Basic medicine
- 3.2 Clinical medicine
Publication Type*
- 3.4. Other publications in conference proceedings (including local)
