Ct value of replicates travel very far - (Mar/02/2007 )

Hi Guys

I am doing real time PCR using various stages of ovary, my trouble is that my CT values travel very far , difference is more than 1 some times, What range of CT is expected , I heard people say 0.6 , I am new at real time , can it be thepippeting error ?? if yes what way I can reduce it.

Thx in advance.
vani

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please give more detail

do you mean your replicates of the same thing are more than 1 Ct value off? or do you mean the difference in multiple samples?

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yeah , they are the replicate frm the same RNA , wht we call as "technical replicates" and I know they should give the same Ct value its like CT 1 = 24 ,Ct2=26

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yeah, that's not so good

how are your dissociation curves? what is your efficiency? do you use SYBR or primer/probe?

how do you control the quality of your RNA prep? (i.e., do you spec it)

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Hi thx for replying,

I spec the RNA , dissociation curves are nornmally ok. and I use syber green, can It be a pipatting error as I am really new in this.
If yes how can I reduce it.
I put the pcr mastermix first, I dilute my RNA in saperate tube thn I mix it in the mastermix.

thx again

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Hey!!

With my experience i would say that the differences in Ct replicates is due to bad pippeting. For my own replicates 0,5 is the acceptable difference. Whem the pipeting is perfect i get difference like 0,01. Check for your tips. Are they really good?!
*

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I've heard O.5, 1.0 and now 0.6. Is there a common consensus in academia, industry, etc? I'm currently working on putting some acceptance criteria Ct of technical replicates in my lab. Anyplace that I can get this information?

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I'm doing absolute quantification of DNA. I found that through months of experiments including dozens of data sets, standard deviations of the known standard resulting in mean ct = 31.74 at efficiency equals 1.832 are 0.25 cycles. Sometimes ct-values are higher, sometimes lower. For me it wasn't possible to do pipetting more exact. Since Real Time PCR depends on exponential mathematics, higher deviations mean significant differing initial template numbers. It depends on PCR amplification efficiency since the formula is N(n)=N0*E^ct . This means that in a PCR with efficiency = 2.0 , a delta ct of 1.0 means double or half the initial template number in one of the samples, while delta ct = 0.5 means a difference factor of 1.41 . The formula is: Factor=E^deltact .

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