standard curve construction/input - specifically on BioRad iCycler (Apr/07/2006 )
Im using a BioRad iCycler. My questions regard
1)inputing standard curve concentrations during plate set up.
2)reading off the standard curve to obtain 'copy number' values.
For example, lets say my dilutions are:
iCycler only lets you input 'scientific notation' which would make those values:
5^-1 (= 0.2 log = -0.699)
10^-1 (= 0.1 )
20^-1 (= 0.05 )
40^-1 (= 0.025 )
80^-1 (= 0.0125 log = -1.9 )
Here is where i am confused. With the iCycler i have been instructed by a collegue to input 'reversed' values. ie. i have been instructed to assign my 1:5 sample as 80^-1, and my 1:80 as 5^-1
My collegue couldnt offer me a satisfactory explination as to why...and as such i was sceptical. Much to my surprise however (which only adds to my confusion) the standard curve came out correctly after the run. It appears that iCycler software read my input of 80^-1 and calculated a log value of 0.699 (which is actually the log of 5^-1!!!!), and read my input of 5^-1 and calculated a log value of 1.9 (which is actually the log of 80^-1!!!). I cannot understand for the life of me what is going on here, and why i need to input 'reversed' values. Furthermore, the log values provided dont have the 'minus' infront of them when surely they should?
Does anyone have experience with the iCycler? I will sleep so much better at night is someone could help shed some light on this for me please?
When using the standard curve to obtain the copy number for a Ct value of interest, the copy number given is actually a 'log value'. To obtain the actual value i am performing an 'inverse log'. Is this the correct thing to do? (For normalisation against housekeeping gene standard curve data etc)
Thankyou to anyone who managed to read this to the end without falling asleep. I hope i managed to explain myself well enough. Thanks very much in advance for any help offered!!!
hi all. It seems that quite a few of you have viewed my post, yet no one has made any suggestions or comments. Are the questions stupid? Do i need to clarify what im asking? Do i need to provide more info? Please let me know what you are thinking and why you are not replying.
i m not very much into this situation. better person who can clear ur doubt would be BIORAD company guys.
I do quite a bit of real time. I have read your post and thought about your questions. however, I just don't know the answers...I work on an ABI machine, I followed the instructions in User Bulletin #2 more or less explicity when setting up my standard curves to get efficiency (quite different from your troubles I think), and I don't worry about copy number I worry about relative levels of transcripts....
I'm sorry you are frustrated! but, this is why I have not helped you; I am unable
I work with a biorad machine, but I input my values in terms of nanograms or picograms, thus the dilution is never an issue.
As for question #2, I am not sure how it works when you use copy numbers, but at least when you use nanograms/picograms as inputs, I believe that the machine calculates the nanogram equivalents of the PCR products directly.
Sorry I can't be of more help.
Thankyou all for your replies. Im still waiting on a reply from BioRad technical guys...hopefully they respond soon. I guess this is my cue to chase them up. Surely these must be standard questions for them. I cant be the only person in the world asking them these questions can i???!!!! Why are they taking so long?!
Im not so knew to real-time. I understand the main and subtle principles etc. I dont even have a reaction problem. My melt curves, standard curves, and realtime graphs all look pretty good. i simply dont understand how to use this particular machine/software. Manual is useless. Technical support have abandoned me. Basically, my data is locked in there until i figure this out. So this is frustrating me enormously. And there is a serious lack of thought or care by a fellow user which is frustrating me even more.
Sorry for the rant. But i feel better for it.