TAQMAN real time - what is this??? (Jun/08/2006 )
Hi to everyone:
I´m doing a qRT-PCR with Taqman probes, I have FAM as quencher and BQH as reporter and the assay I´m attatching is the second I have done but I have no idea what this kind of curves mean.
The curves start in negative values, start to rise and then fall. What is that??
The green curve has 2 microliters of RNA (extracted with a silica gel column method) and is the only one that has a Ct, the primer concetration used is 100/100 nM. The red curve is the same but with a different primer concentration ( 200nM/200nM).
The grey line has a dilution of 10 -2 and 200/200 nM of primers and the blue line is my no template control!!
The concetration of my probe is 200nM
I´m completly lost, I really will apreciate any help! 
So....are you doing one-step RT-PCR? or is your '2 ul RNA' your no-RT control? that will explain a lot
why do you say only the green has a Ct? they all have Ct's
the fact that your green and grey lines are two Ct's apart is good; your dilutions are spot-on and your pipetting is good
the difference between the red and green shows what happens when you add too much primer (I really think the green looks the best)
two other things: 1. why aren't you doing replicates? 2. I would back off template amount a little. it may make the assay more sensitive for you
Hello again:
Thank you very much for your fast reply but I still have some doubts
I´m doing one step RT-PCR and the primer concentration is low ( I think: 100nM) but I will follow your advice and try with less concentration.
Other thing: my grey line is my no template control, it has no template at all and it has fluorescense at the begining. I don´t do no RT control.
I did replicates but they didn´t have the same aspect as the one I showed on the graphic so I didn´t post them.
When I say the lines don´t have Ct is because the Mx3000 software says they haven´t and I think is because they rise but then they fall really fast.
My big big question is: why the curves fall down?? Is that saturation or maybe the probe is not well designed?? I designed the probe and primers with Primer Express, wich I think is a good program, and chose the best pair (the one with less penalty score)
I will be really thankful if you answer this questions 
Than you!!
why do you say only the green has a Ct? they all have Ct's
the fact that your green and grey lines are two Ct's apart is good; your dilutions are spot-on and your pipetting is good
the difference between the red and green shows what happens when you add too much primer (I really think the green looks the best)
two other things: 1. why aren't you doing replicates? 2. I would back off template amount a little. it may make the assay more sensitive for you
It seems like your baseline setting is set up from 3 to 15 and you have curves before the 15 cycle. Try to set up from 1 to 2 and look if now they seem like real amplification curves......
Micky
So....are you doing one-step RT-PCR? or is your '2 ul RNA' your no-RT control? that will explain a lot
why do you say only the green has a Ct? they all have Ct's
the fact that your green and grey lines are two Ct's apart is good; your dilutions are spot-on and your pipetting is good
the difference between the red and green shows what happens when you add too much primer (I really think the green looks the best)
two other things: 1. why aren't you doing replicates? 2. I would back off template amount a little. it may make the assay more sensitive for you
It seems like your baseline setting is set up from 3 to 15 and you have curves before the 15 cycle. Try to set up from 1 to 2 and look if now they seem like real amplification curves......
Micky
i agree with micky. the only time i have seen curves fall off like that is when the baseline values need to be played around with because we have used too much template.
you could also run out your products on a gel to determine you are getting the correct amplicon size. use agarose 1000.