The MCF-10 cell line was grown with DMEM12, 10% serum, 5% antibiotic and HuMEC supplemented. When we studied gene expression of LKB1 by RT-pcr ROCHE the results is small in triplicate (CT 35 ,42 .48 ) (each CT has a different melting curve although all of them for one product) with primer lkb1 but with primer of GAPDH the CT value is 11.15.
The sample dilution is 1:5
Asma, It is normal for one product to have different melting curves in triplicate because of unavoidable sample-taking errors, especially for genes encoding low abundance proteins, however for the internal control GAPDH, encoded by a house-keeping gene, a little error does not make a difference.
It is possible that the lack of regulation by LKB1 is part of what makes it a cancer line; AMPK activation by LKB1 suppresses proliferation, so maybe MCF10 cells have constitutively low expression anyways. As it is a 'nutrient' sensor, you could try additional conditions with your media; high vs. no glucose, 10% serum vs. 0.5%...
I'm sure you already went through the step of double checking your primers?
I think everett gave a key hint for you.
are you sure that your primer pair is the best suitable for the detection? if you did not make test for efficiency, you should. applying primers to serial dilutions of the template. you can follow, for examples, guidelines as described in Schmittgen and Livak (Nature protocols, 2008).
The loss of Lkb1 gene is important to overcome oncogene-induced senescence (OIS). OIS is a tumor-suppressor mechanism for the carcinogenesis, because ongoenic molecule and aberrant activated survival/ proliferation signal pathway leads to senescence with up-regulation of p16, p19, and p21, all of which belong to cyclin kinase inhibitors and are responsible for cell cycle arrest. It has recently been reported that BRAF(V600E)-induced OIS in primary melanocytes is eliminated by the synergistic depletion of both CDKN2a gene, encoding p16 and p19, and Lkb1 gene, the negative regulator of mTOR signal pathway (Cancer Cell Volume 27, Issue 1, p41–56, 12 January 2015).
thank you for all
i was check the primer before ..
also .mcf-10 is normal cell and have low expression lkb1 and i have mcf-7 cancer have more gene expression of lkb1 that different result from other studies
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression