Hello Tanya

I study gene expression in human macrophage. I use PIK3C2A as housekeeping.

Please, check the articles:

https://www.researchgate.net/publication/322944525_miR-505-3p_controls_chemokine_receptor_up-regulation_in_macrophages_role_in_familial_hypercholesterolemia

Article Macrophages of genetically characterized familial hyperchole...

LDL accelerates monocyte to macrophage differentiation: Effects on adhesion and anoikis

Best regards

Hey Tanja,

we also perform qRT-PCR with BMDM (naive or after LPS treatment or bacterial infection) and we used actin as housekeeping gene.

For the genes we investigated it worked well, but I do not know, if it fits for your setting. I would also recommend to check not only qRT-PCR but also protein levels of your target protein via Western Blot, because mRNA levels do not always correlate with protein levels.

For an example of a pRT-PCR and the corresponding Western Blot (methods and results) please have a look at Supp. Fig. 7D in our paper:

Gluschko A, Herb M, Wiegmann K, Krut O, Neiss WF, Utermöhlen O, Krönke M, Schramm M. The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes. Cell Host Microbe. 2018 Mar 14;23(3):324-337.e5. doi: 10.1016/j.chom.2018.01.018.

Please feel free to ask any further questions.

All the best and a happy new year,

Marc

I agrree to the aforementioned comments. However, it strongly depends on your treatment which gene is not affected under specific conditions and may be - therefore - used as "housekeeper/control". This can be tested by try and error methods.

I recommend you the database "Housekeeping and Reference Transcript Atlas" (www.housekeeping.unicamp.br) for candidate reference transcripts. It offers tissue/cell (including macrophage) selective reference transcripts for qPCR normalization. Interestingly, some validated ready to use primers are also available in this database.