In my study on human tumors, samples showed down regulation of one of my genes of interest. I am interested to know whether its because of promoter methylation or transcriptional inhibition. How should I go about it? I to have check for tumor cell line, but they don't express this gene. How can I find out the reason?
In case to understand about your promoter methylation or demythelation you can use Bisulfite genomic sequencing .
Hi Zhi Xu,
yes almost 70-80 % of my tumors show down regulation, and regulation of this gene is not know yet, aCGH data sugessted that this down regulation is copy mumber independent thats all detail know about it,,, so i dint understand where to start.
there may be many factors...one is as you tell methylation of promoter sequence.....another may be unstability of mrna.........of that gene ......
there may be many factors...one is as you tell methylation of promoter sequence.....another may be unstability of mrna.........of that gene ......
If you want to check, if the transcription is inhibited, you can perform CHIP to see, if the DNA Polymerase is bound to the gene of interest, promoter and ORF.
can u tell me how to check promoter CpG by gene sequence or any specific online tool
Use online tools (e.g. NCBI Gene or GenBank) to find the gene directly upstream of your target gene. Use a forward promoter for the last 10-15 nucleic acids of that upstream gene and a reverse primer for the end of your gene of interest; perform PCR; run the results on a gel (Southern blot). You may end up with two results:
a.) two fragments appearing in your gel: one being the last 10-15 nucleotides of the upstream gene of your target, with or without some CpG islands, if they are present (likely being the shorter, lighter fragment), and the other being the promoter of your target gene plus your target gene, with or without some residual CpG in and around the promoter, corresponding to the most downstream methylation site (this likely being the longer and heavier fragment). (There is also the miniscule chance you will also get a full-length fragment of the end of the upstream gene, CpG islands, promoter and target gene, on the off chance only one side of the DNA strand is methylated). - or -
b.) a single fragment in your gel corresponding to the full target gene with promoter and CpG islands (if there are any).
If instance a.) occurred, you have methylation of CpG islands. If instance b.) occurred, there is no methylation, so you next step might be to run RT-PCR to find the number of mRNA transcripts, compare to normal cells. Alternatively, you could genetically engineer the cells to carry a reporter luciferase enzyme at the downstream end of the gene and a quencher upstream of it, then perform fluorescence microscopy to see when/where it becomes degraded in your cells. You may also want to check the sequence of your target gene for any mutations that might have spawned a premature stop codon.
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