# What is the optimum region of the curve ? - (Jun/10/2008 )

Hi

What is the optimum/best region to analyse the curves (PLACE THE ORANGE LINE) of my real time RT-PCR?

is it at the begining where the curves come up rapidly (BOX b- THE REGION DENOTED BY THE BLUE LINE) or when they seem to be linear (BOX A- THE REGION DENOTED BY THE RED LINE)?? Shown in the graph.

Will it make a big difference for the results??

Thanks

[attachment=4817:linearpc...ction2_x.jpg]

What is the optimum/best region to analyse the curves (PLACE THE ORANGE LINE) of my real time RT-PCR?

is it at the begining where the curves come up rapidly (BOX b- THE REGION DENOTED BY THE BLUE LINE) or when they seem to be linear (BOX A- THE REGION DENOTED BY THE RED LINE)?? Shown in the graph.

Will it make a big difference for the results??

Thanks

[attachment=4817:linearpc...ction2_x.jpg]

**middle**of the

**middle**third. Not the beginning, nor the end of the upward slope.

this is the linear view of your plot. you cannot set the treshold in the linear view. you have to change to the log-view and you should set the threshold in the linear phase of the log-view (this is the exponential phase of your amplification). The exponential phase in the linear view would correspond to the region where the orange line crosses your curve.

Dear All ,

Thanks for the replies.

This is why I am confused. different people have different opinions about the best region to take the measurement.

Please Cell-counter can you define the region from the values from the Y axis.

Hi Ned land thanks for the reply. In my lab we have never done this (turning the graph in to log view). could you please describe in detail how you do it.

Thanks in advance

hello, i have attached a document where correct baseline and treshold setting is explained for a ABI 7700 machine, but the fundamental rules apply also to other real time machines.

if you are new to real time pcr i recommend to go to the Applied Biosystems homepage and look under "support". you will find many useful documents there.

https://www2.appliedbiosystems.com/support/apptech/#rt_pcr2

Thanks for the replies.

This is why I am confused. different people have different opinions about the best region to take the measurement.

Please Cell-counter can you define the region from the values from the Y axis.

I think Ned has already given you best advice and links.

pcrmossad,

It looks like you have the BioRad Icycler? If this is so, why not let the auto calc do the work? I found some info in pg 63-66 of the iCycler iQ Multicolor Real Time PCR detection system Operating instructions. Cat # 170 8740

It looks like you have the BioRad Icycler? If this is so, why not let the auto calc do the work? I found some info in pg 63-66 of the iCycler iQ Multicolor Real Time PCR detection system Operating instructions. Cat # 170 8740

yes I use i Cycler , but single colour. I can use the auto detection , however it doesn't always help to get a good standard curve. anyway I will go back and read if i missed anything. Thanks!