18S Melt curve - sorry for the cross post.. this forum gets more traffic. (Dec/21/2006 )
I've been doing qPCR for about 5 years, but recently I started in a new lab and have (happily) changed model systems (i now work with T-cells; previously I was working in a yeast lab). For a long time I've used 18S rRNA as an internal control (it pwns the others IMHO), so when I started doing qPCR with RNA samples taken from human cell lines, I redesigned my 18S primers for the human 18S gene(s) and did all the standard melt curve analysis, controls, etc. I'm using 5ng of cDNA per qPCR reaction. My 18S rRNA CT is around 12-13 and my target gene is around 25 - 30 CTs (depending on the conditions).
I see only 1 band on my post-qPCR gel (two concentrations of cDNA template; click the thumbnail to see the full size).
but when i do melting curve analysis i get this odd LONG shoulder on the left before my usual peak. Click the tuhmbnail below for a full size image.
What do you make of this? I searched all of the internet and could not find another melt curve for qPCR reactions done with 18S anywhere (even from companies which sell premade 18S rRNA primers).
Also, My CT values are linear over 8 logs of input cDNA concentrations. Also, when I do a no-RT i get CT values of around 39-40 or nothing at all. and a NTC control doesnt amply anything.
Before you answer.. here's what i think, probably becuase i previously came from a lab that studied translation and ribosome structure function: since 18S rRNA has so much secondary structure, the odd melt curve may be due to secondary structure present in the amplicons themselves (since the reactions had previously been run for 40 cycles).
Is this melting curve from you standard?
What is the initial cDNA concentration you added into your reaction?