I am using a Biorad real time pcr device which is quiet old and cyber green method. I have made different dilution twice (once dilution factor 2 and second time 5) four series from first to the last one they have been decreased.but the problem is that my third dilution's curve rose sooner than the forth(dilution factor 2) and I reaped it with dilution factor 5 now the second and first have a same problem. do u think my primer has a problem.
Hi there. Could you post a picture of your standard curve? How far off from the expected Ct are those points? If a higher Ct is found for the first (more concentrated cDNA) points of your curve, it probably means PCR inhibition. What is the initial amount of cDNA are you using to do this?
I attached the word format of my curves. The first one that has been rose is my second dilution
Hmmm your data is a bit confusing...
In the first graph, you did each cDNA dilution in duplicates, right? If that's the case, I only see 4 dilutions there. And the duplicates are not good. The scale in the Y-axis is linear, you should switch to log scale because it is easier to see the inflexion of the curves to establish the threshold.
Second graph is the melting curves. right? Based on the peak sizes, I'm guessing the dark blue curve is your more concentrated cDNA, cyan and black curves are the first dilution ... then I can't distinguish the rest.
The linear regression graph looks very confusing to me as well.
How did you design this experiment? Ok the initial amount of cDNA was 4uL, but what is the TOTAL amount of it in each dilution, in ng ?
This is how I design my experiments to determine PCR efficiency:
A) Serial cDNA dilutions
Total cDNA amount in each point:
First point: 100 ng/uL, then make serial dilutions of this cDNA 1:5, total 5 points. I then use 2uL for each reaction (2uL cDNA + 12.5 SYBR Green Master Mix + 2.5 primer F + 2.5 primer R + 2 water = 25uL total volume).
So I'd have, in total cDNA amount, for each point::
1- 200ng cDNA
2- 40ng cDNA
3 - 8ng cDNA
4 - 8/5 ng cDNA
5 - 8/25 ng cDNA
In duplicates. You must also have a negative control (water instead of cDNA) for each point
B) Perform qRT-PCR reaction and melting analysis. Establish threshold. The software probably does that for you, but I saw it is detecting the baseline (noise) at cycle 18 in one curve, so you should set the threshold yourself.
C) Plot log(cDNA amount) x cycle threshold (Ct), do linear regression. Each duplicate mean should be distant around 2.3 Cts from one another, if your primer efficiency is around 100%.
D) Get slope and calculate Efficiency E=10^(-1/slope). I'm guessing the software does that for you as well.
i am using biorad i-cycler mashin
and in my total reaction mixture i have (10 cybergreen biorad +4 cdna+5.88 water+0.06 primer(f and R)=total 20).and i am runing it in triplicate.and triplicate blank for all of them.
in the pic i have sent you the dark blue curve is for my second dilution(more diluted )and it has been rose first.
the other problem is that
i have a big variability in each dilution and i don't obtain separated and distinct bunch of curves for each dilution as i used to take in other primers such as my house keeping gene
r u using also biorad device?
Hmm ok, maybe something went wrong when you diluted your cDNA, have you tried doing another series of dilutions and repeating the qRT-PCR? I'd also watch out for the threshold, as, like I said, it is detecting stuff that it isnt't supposed to detect (e.g. baseline at cycle 18).
I use an ABI LightCycler (a bit old too), but it works well
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