Dear Anna,

After looking to your doc file, it seems to me a problem in dilutions series. The strange values in amplification efficiency as well as in R^2 may be surpassed in you perform a new dilution series, since sometimes template diluted degrades due to frost/defrosting events. Also, in your doc file all quantities in podoplanin gene are amplified more and less on the same cycle threshold, ranging from cycle 20 and 21. Despite having a specific melting curve peak and an electrophoresis expected product, amplification efficiency is not related to this features. The main considerations rely on dilutions series fresh, well-performed and absence of PCR inhibitors.

Hope this helps.

Filipa

What is the cDNA quantity in ng (RNA equivalents) in per qPCR reaction in the more concentrated samples ? Have you tried diluting further your cDNA?

Having looked at the attachment , It seems to me some kind of inhibition is occurring. Besides, E>>>100 is a good clue for inhibition. Too much cDNA could be inhibitory and this effect could be amplicon dependent, which could explain why everything seems with with ACTB and not with your other amplicon.

Did you amplify both genes using the same dilution series? Can you tell us what your dilution series was?

quantity of RNA was 1ug for both templates (different cell lines). Dilution series was 5 fold, prepared the same way for both genes.

thank you for your anwsers,

Anna

Have you repeated this? Your data suggests that you have only amplified one concentration. It would seem odd that inhibition would be the issue since you have a nice solid Cq value- doesn't look too high or too low. I would repeat your dilution series making sure to vortex the cDNA before pipetting and use low retention tubes if possible. One comment about terminology- you mentioned the E was really high. You actually cannot make any statements about E because your dilution series failed, so an E calculation is not possible.

Beside creating a standard curve, you can use the software LinRegPCR for calculating the efficiency of every single reaction. It is freely available in the internet. I agree with Karen, that the curves look actually fine in terms of efficiency and Cts. And I also agree that you may repeat the dilution series.

Anna, do you mean that the first qPCR reaction from the serie for both genes received 1 microgram cDNA , the second 200ng and so on ?

If that's the case, you put a way too much of cDNA per reaction.

I agree with Vivian, the LinRegPCR can be used for qPCR efficiency estimation from a single reaction (I'm using it and am a big fan!), but nevertheless if such a discrepancy is observed at the "golden standard", LinRegPCR will also not estimate the E correctly.

Dear Anna,

did you have NTC and no RT controls? Where they okay?

Dear Petar,

Anna wrote that she had used 1µg of RNA (I guess in a one-step PCR?), not cDNA. I think that amount is fine?

Of course, estimating the efficiency with LinRegPCR does not solve the problem of having nearly the same Ct for all dilutions. But with the "standard curve" that is shown in the file, no meaningful efficiency can be calculated. I think, the software should at least give more hints to find the problem. For example, if there is inhibition, one would expect a higher efficiency in the last dilutions than in the first ones, don't you agree?

I completely agree Vivian,

inhibition usually "flattens" the standard curve at higher concentrations, the Ct of the technical replicates are also very different, but with the further dilution points the replicates become closer and the curve - normal. I'm attaching a figure from "Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR "

Regarding the RNA/cDNA input per one-step/two-step qPCR, I guess the maximum tolerable amount per reaction depends mostly on the master mix and the polymerase in it (and the RT kit). With the reagents I use I found that most of my assays are linear between 1:10 to 1:400 dilution of cDNA concentrated 20ng/microliter (e.g. 2 to 0.05ng per reaction). But you're right that that values cannot be extrapolated from place to place.

And oh yes, LinReg definitively is very useful for comparing efficiencies, when all different dilution points are made up of the same master mix.

From the attached data I see both internal controls are a problem. although the alpha actin run is good the duplicates are not tight, since the std curve gives good numbers in term of slope and intercept you can use if this was the only control determinate in unknown runs. If both the runs were made with all things exactly same I mean cDNA, pcr run mix, pcr conditions, machine. the problem of the cDNA prep is clear in the second internal control podopline. number one, the concentrations of cDNA does not work well at all, all the amplication is at cyle 20 or 21. First thing to do is to test run the cDNA with just three concentration, lowest highest and medium concentration for all the internal controls you choose at the same time with all same conditions in the same pcr run, once the test runs go good then extend the concentrations above the low and below high the first test runs, the results are invariably great and unbelievable if you are meticulous and taking care of each step. Good luck

From the attached doc file I would think that you have a considerable contamination with podoplanin. What do your no-template-controls and no-RT-controls look like?

Btw: never ever even think about using data like the one shown for podoplanin to calculate efficiencies. There is absolutely no sense in calculating E from such data. The statement that "E is low" is not just wrong, it is misleading. Calculate the confidence interval as well and you will see what I mean.