using a diluted sample is acceptable, but keep in mind that it is a RELATIVE standard curve not an absolute. meaning it can tell you how much more is in one sample versus another but can't tell you # of copies of target. you could order a synthesized RNA target, convert to cDNA and use that known concentration for an absolute standard curve but synthesized RNA that long can be expensive.

Julie Ann Dougherty Thanks for the answer! Yeah right now I'm using the relative standard curve method as I dont have any endogenous control to do the absolute one. I was just wondering if there is actually a rule for what kind of samples that we have to use for standard curve, especially to find an amount of spiked sample. Since my spiked sample is a DNA extracted from a mixture of meat, primer targetting porcine DNA, it just occur to me that to quantify the DNA do I need to use standard curve from raw sample (pork) or is it okay for me to just use that diluted spiked sample.

So far, I'm just using the diluted spiked sample for standard curve since I saw some studies doing it but idk, i hope i didnt interpret it wrong.

Also, based on your answer since Im using a relative standard curve does it also means that we have to run a standard curve each time we run a qpcr, even if we want to find the same target DNA?

Metty Ariani

using the diluted spiked sample should be ok as long as you are just looking for an increase or decrease and not an absolute amount. You don't need to run a relative standard curve for each qPCR, but it could be a good plate control to always run the same sample on each plate. the relative standard curve tells you the efficiency of the qPCR reaction and if you are getting appropriate amplification.

see last page of this for "Plate 2: Verify Assay Efficiency with a Standard Curve"

Thanks for the answer and the paper! I wasnt really sure before whether I need to run standard curve each time I do the qpcr or not, and since the amount of wells is not enough and thus I need to use different plate for another type of samples (different treatment, but still same target DNA). For qPCR efficiency, should they generate a same or similiar value? Cause I always got a different one each time I run them

Julie Ann Dougherty Never mind I decided to just ask this in a different questions. Thanks for answering me again!

Metty Ariani you're welcome for the help. the efficiency should be the same every time, this does not mean the Ct's are the same but that the slope of the log10 of concentration on x-axis vs cycle # on y-axis is the same

Julie Ann Dougherty Thank you for your explanations! It really helps me!! I'll further into my experiment then

Julie Ann Dougherty Hi again! I'm so sorry to bother you. This might be a dumb question, but can we use the same source of positive control sample for the standard curve and unknown sample? I read this on this one:

https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6894.pdf

Heres what I do: I checked my positive control and then diluted it for standards, but also put them in unknown samples without diluting any of them.

And they said they generate standard curve from positive control.

I honestly kinda confused at first on why do we need unknown samples since basically we could knew their concentration from nanodrops, but I guess its because nanodrop couldnt really tell us whether its a DNA or not and by using qPCR, its more accurate. But what do you think?

for the standard curve you can use any sample that you know has target in it. You are using the positive control DNA as a spike-in to the unknowns? then this is fine to use as well as long as the melt curve shows only one product amplifying. The nanodrop tells you the concentration of all of the DNA in the tube not just the target DNA this includes degraded or contaminating DNA (except if your positive controls if those are synthesized DNA). if you are using a positive control that is only the target DNA then you can measure the concentration by nanodrop and use that as an absolute standard curve to determine the amount that is spiked into the unknowns. i'm a little confused on the experimental set up. Running a standard curve tests the efficiency of the primers/reaction, it should be doubling every cycle. sometime factors inhibit or promote the reaction to make the efficiency lower or higher. this is why you should aim for an efficiency near 2.0 (1.90-2.05)

Julie Ann Dougherty Thank you so much for explaining it to me! I think I did it just the same like you said. However, I am using the positive control DNA as a spike-in to the unknowns instead of positive control that only contain the target for standard curve. And since I'm using a taqman, I'm afraid I cant use melting curve and maybe my options left will be through gel if I want to confirm it. Thanks again for taking your time!