I had mcf-10 cell and was grown in dmem/f12 media with fbs and AB and rna and dna was extracted from the cell ,, when I did RT-PCR with cDNA , I got results with GAPDH but not with my interested gene. Although I tried different conc of cDNA , all of them had no amplification ..and the primer works with other cell line .. ..
Are you sure this gene is expressed in these cells? This is a bit obvious, but not all genes are expressed in all cells at all times. Alternatively, the cells could have a different splice isoform that doesn't contain part of the primer sequence.
Other alternative explanations include bad quality starting RNA. If you are using oligoDT for your reverse transcriptase reaction, if the RNA is fragmented you probably won't amplify anything thats far away from the polyA tail. Also, if the product sequence is much longer than the GAPDH, the RNA quality is a factor even for random hexamers (which you should use for small product sizes anyway).
You should also check the RT-PCR of your "other" cell line for efficiency and product size to be sure you are amplifying the correct product or getting primer dimerization or an off target. Lastly, if your product sequence does not cross an exon-intron boundry, your "good" reaction in the other cell line could just be amplification of genomic DNA. Consider using DNAse before the RT reaction. I am have not used these cells before or looked at the gene, so I am sorry I don't have any more specific advice.
Maybe your cells do not have your gene of interest? An other explanation could be that your primer is not good and finds some unspecific products in your other cells. Did you already check the meltingpoint of these products? Besides that, how did you designe your primers? I use Roche probe library and it works very good!
The easiest answer is that your cells has not the gene that you are interested or they have it in a very low expression rate.
Have you tried different melting temperatures?
Good luck!
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