Do I need internal controls in MS-PCR? - Methylation specific PCR Internal controls (Jan/10/2006 )
I have set up an MS-PCR assay to determine if CpG sites are methylated in brain tumours. Do I need to have an internal control in my assays? If not how do I know that the samples not showing any product in the meth PCR are truly unmethylated and not just failed PCR's?
Does anyone use housekeeping genes as internal controls and do mulitplexed MS-PCR's work OK?
indeed you would need internal controls to check if your PCR is working and if indeed your conversion was efficient.
as for what controls, that is up to you, I don't think houskeeping genes would help because they are consituitively expressed and there was a CpG island associated with it, it would be invarably be hypomethylated.
I would suggest you make your own. use either genomic DNA or a plasmid construct with your gene of interest and SssI methylate the DNA so it's fully methylated and then run it alongside with your samples.
Using genomic DNA from a female should work well as a control - as they always have CpG sites.
that's a great idea actually jan,
also you could direct primers to imprinted regions as well as these are differentially methylated in normal patients