I am using 5-aza-2′-deoxycytidine to check promoter methylation of a gene as litreature say so ,but after RT-PCR the expression of the gene was decreased , what dose that mean ? could be due to toxicity?
Dear Abrar,It seems to me that the easiest way in this situation is to try 5-aza from another vial, but you should also properly check the procedure of a method you use. Moreover such results may show that 5-aza induces expression of a strong transcriptional repressor of a gene of your interest. IMHO, you'd better try one more methyl transferase inhibitor, it may be a related to 5aza compound to compare results.
I would pay attention to your normaliser. E.g. if you use GAPDH or similar housekeeping gene to normalise the expression levels, GAPDH may be more expressed after 5-aza treatment leading to (false) down regulation of your gene.
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