3 replies to this topic

[rajah]

Hi guys,

just wondering, which quantitation method do you think is the best to use in relative qPCR? Standard curve method (I wonder if someone prefers this because it´s so laborious with standard dilutions on every plate), Pfaffl method (with efficiency determined on several different occasions) or the deltadelta CT method (which I hear is been criticized to be more of an approximation)? I´m just choosing the method to use; I was advised to use the delta method, but after reading several papers I´m starting to think Pfaffl would be better. Please let me know your opinions and experiences!

Also: when determining the efficiency, can I just use a dilution of f.e. some of my actual samples? or as I use brain tissue samples, can I determine the efficiency of my primers in a sample from another part of the brain (from which I don´t determine anything and is then "waste")? *get the idea of determining the efficiency but apperently not quite getting the realization *

Thanks alot in advance!!

Edited by rajah, 07 April 2009 - 05:26 AM.

[molgen]

I use the Standard curve method.
First, it's not that laborious.
Second, you know the efficiency this way.

You're better off using the same tissue for the efficiency test as for the experiments.

[Dr Teeth]

molgen, on Apr 7 2009, 10:17 AM, said:

I use the Standard curve method.
First, it's not that laborious.
Second, you know the efficiency this way.

You're better off using the same tissue for the efficiency test as for the experiments.

I've used both delta delta Ct and the Pfaffl equation.  Unless the amplification efficiency for your target and control differ by more than 10% (at which point, you should design alternate primers anyway), then I obtain nearly identical results with either method.  While the Pfaffl method is technically more precise than delta delta Ct (the approximation), results are identical when both primer sets have efficiencies of 100%.  Therefore, we test our amplification efficiencies a few times to ensure that our primers are good and then proceed with the ddCT method.

In short, design good primers with efficiencies near 100% and use any method you want!  
Also, Molgen is right, use the same tissue to evaluate your efficiencies.

Edited by Dr Teeth, 07 April 2009 - 08:39 AM.

[littleaxt]

Hi!

I agree, Pfaffls method is just the efficiency corrected delta-delta Ct method, therefore it is more precise, when your different primer systems have different efficiencies. So it seems more important how you assess you reaction efficiency correctly (standard curve, linear regression, second  derivative, curve fit...).
I'd like to hear some firt-hand experiences :-)

All the best!

Jan