Dilution series between HKG and GOI - (Apr/23/2014 )

Hello all,

I want to ask something about my qPCR assay, it's absolute quantification, and I ran the HKG first. And for the standard, I made dilution series with dilution factor 1:5. The standard curve came out good, but when I ran the standard for GOI, 1:5 dilution factor didn't come out well, so I did 1:8 dilution series, and it went good (the efficiency percentage reach 100%). So I wanna ask, is that okay to run a different dilution factor between the HKG and GOI?

Thank you.

Cheers,

detriar

You use dilution only for efficiency calculation?

Well then obviously the undiluted concentration is already inhibiting the reaction.

Do you care more about what efficiency do you get with dilution series, of what actually is your reaction efficiency in the set range?

Dear , thank you for the response but I'm sorry I don't think I get your questions, I had a terrible way to understand English , so I'm gonna explain as good as I can.

I was told to pay attention to the slope, and efficiency percentage in absolute qPCR. So when my 5-points of standard did not get the efficiency above 95%, I try to do more. I was told if 10-fold dilution factor does not work, I have to try 4 or 5-fold dilution factor for that 5 points.

For the cDNA samples, between the GOI and HKG there are no differencies in amount, so I can say that the samples are all the same. It's just differency on the dilution factor of each their standards.

Thank you

Trof on Fri Apr 25 16:33:33 2014 said:

Do you care more about what efficiency do you get with dilution series, of what actually is your reaction efficiency in the set range?

I must admit I also wondered about this, because I thought that it's OK to try a different dilution if for instance the resulting curve is not linear and you want to get a more reliable value for the effciency. A colleague of mine did so once. Is something wrong with that ?

The efficiency is actually a property of each reaction alone. But it's hard to calculate from the single one (there are tools to do that, from each amplification curve, but they are not commonly used).

So in theory efficiency should be constant within dilution series and from several known concentration points you can calculate it.

So, imagine you use 1:5 dilution and get a bad efficiency. It's known that non-linear and/or too high efficiency means that some concentration points have different efficiency. Usually the least diluted one is inhibited or something. 

So, within that series you use (i.e. undiluted cDNA and dilutions) it means, probably that the undiluted is "screwed".  You use 1:8 and now it looks good. Does it mean now that the undiluted is not inhibited or something any more? Of course it doesn't. 

By making different dilutions of the original, you are not adressing the problem (unless the problem was bad dilution in the first place, that is also possible) but only the output.

Of course, from a non-linear dilution curve you can't (usualy) calculate efficincy at all. So to get at least an idea, you need to make some linear. BUT.. that still means some of the points from the original one are amplifying with different efficiency than others -> leads to different extrapolations of the initial template concentration.

That's why the rule should be that if your dilution curve is linear and spans a defined range, that all samples of the same kind within that range have this defined efficiency. Outside that.. you don't know.

 So making several dilutions until you get the one that "looks good enough" is not solving anything. Just hiding the "bad" out of sight.

It's something like for example "fixing" the too high number of patients that died on your clinic, by moving those about to die outside to the hall. They will be dead anyway, but your numbers will look better.

Oh OK, I understand. Pehaps my colleague did it just to make sure the dilution was not bad in the first place, as you said.

Trof on Sat Apr 26 22:06:45 2014 said:

The efficiency is actually a property of each reaction alone. But it's hard to calculate from the single one (there are tools to do that, from each amplification curve, but they are not commonly used).

So in theory efficiency should be constant within dilution series and from several known concentration points you can calculate it.

Dear ,

actually, because I got curious about this, I re-assayed my standard, using 1:5 dilution factor. And the result came out quite good, I got slope -3.37. The first 1:5 dilution series, I think I made a pipetting mistake, or other errors.

Now I can use the same dilution factor between the HKG and GOI. Thank you.

detriar: Nice. That is also an important thing to know. If you get repeatedly different results with the same dilutions, you know there can be something wrong with the other pipeting you do as well. It may be only technique but also uncalibrated pipettes. It's worth monitoring too.