Product with different Tm among samples - Sybr Green gives products with differing Tm of ~2-3 degrees (Feb/14/2007 )
Any suggestions on why this would happen? And if this changes my gene expression data?
This is one gene with same primer master mix for several different samples.
Strange... Did you check the product sizes on a gel?
in the case of sybr green any two melting tms with a difference of more than 0.5 degrees are not the same samples. Infact, that is the basis of diagnostic criteria for screening samples for infectious diseases using commercial kits. I'm giving this ex. only if you are using the Smart Cycler system and sybr green.
I think that theses samples have a different sequence, that means that are different things. Check them on a gel and sequence them.
a couple things come readily to mind:
1. your primers could be croaking. get a new set and see how they look
2. there is contamination
3. there is template degradation, at any of the steps, and you're possibly getting truncated versions of product
4. how is your efficiency?
Could you just tell us, what samples you are working on? We had the same problem with the SH-SY5Y cell line - only thing that helped was designing new primers... and we had already hoped, that we have found some new splice variants of the gene
I really think this is what you see when you are looking at samples from different patients with different genotypes... You probably have the right amplimer there are just different SNPs in different people... Run on a gel to confirm is the right size amplimer then clone and sequence a couple to confirm if you want to be sure...
ah....having never worked with SNPs, I don't think of them readily but that sure makes sense