Running real time pcr product on gel?.. - (Sep/20/2010 )
Hi everybody,
I have a doubt,hope this forum could clarify me better...,i ran a real time pcr and estimated the relative expression using dct method, i got some 14 fold difference between two samples,my question is, is it right option to see this difference in expression on running the real time product on Agarose gel? i even tried,i could sense some difference visually but i dnt think it is 14 times. so i dnt know whether i can believe the RT data,or the visible fluorescent band i saw on gel....anybody pls clarify my doubt. awaiting for ur response.
Gnana...
GNANA on Mon Sep 20 16:02:23 2010 said:
Hi everybody,
I have a doubt,hope this forum could clarify me better...,i ran a real time pcr and estimated the relative expression using dct method, i got some 14 fold difference between two samples,my question is, is it right option to see this difference in expression on running the real time product on Agarose gel? i even tried,i could sense some difference visually but i dnt think it is 14 times. so i dnt know whether i can believe the RT data,or the visible fluorescent band i saw on gel....anybody pls clarify my doubt. awaiting for ur response.
Gnana...
The gel actually referrs to the end of the pcr reaction (hence, end-point or conventional pcr). The end of the reaction is prone to stochastic effects and unrealiable in the context of quantitation. On the other hand, Ct values are calculated on the basis of early exponential phase.
oh s,,,,this makes sense,,,thanks a lot.....then another question i could see amplification in -RT (ct >30) sample and NTC as well rarely, probably it s primer dimers, my question is does the primer dimer formed in -RT or NTC has any influence over the ct value got in the experimetnal sample with same priemrs. when i run the experimetnal and the -RT on gel i could see the primer dimer only in -RT control but not in the experimental cDNA...if this is the case can i still believe the data?
thankings,
Gnana...
Are you using probe-based assay or SYBR Green assay?
If you get amplification in NTC in a probe assay, dimers are unlikely. If you're using SYBR, then you can add melting step on the end of amplification to see if you get Tm of the NTC on the same temperature as your experimantal cDNA. If its like 10 deg lower, then it is dimer. You can then check for secondary dimer peak in your experimental samples, if there is none, all fluorescence is from your specific product and you can use the data. If you get the same peak as in NTC in RT- control, then it's probably both dimers and not DNA contamination.
There is something called template-dependent primer dimers, that will only get dimers in the reaction without template, I've seen a lot of those.
When you see your dimers on the gel in RT- only (and NTC, did you run NTC on the gel as well?), then it is likely, it's only template-dependent dimer and you can use the data, but melting curve is be more sensitive (you may not see the band on gel, but it can still be present).
Thanks a lot trof,,,ur explanantion is really very useful for me..
am using SYBR green assay. on reading ur detailed expln it sounds like mine also is a template dependent primer dimers, anyhow i ll analyse the melting curve again and then i give the data to my prof...i checked on gel both the NTC and RT-, in both i saw Pimer dimers but not in the experimental samples. you have solved my this issue. thanks again.
Gnana...