If you have a R2 value of 0.998 but a E value of 130%, something is wrong with your E. Generally, this would not be acceptable (reviewers will point at it). Some people, indeed, think that R2 value may be important, however, it doesn't really say much about what is happening in your experiment.

In addition, I just really want to say that neither E nor R2 is "accurate". Accuracy refers to the closeness of a measured value to a standard or known value.

https://labwrite.ncsu.edu/Experimental%20Design/accuracyprecision.htm

In RT-qPCR THERE ARE NO KNOWN Efficiency values. You don't, and you never will know what the "true efficiency" of your primer-DNA combination is. Because the standards don't exist. For example, you can know what "true 1 meter" is because there is a human definition of that, and how much it should be, and a standard exists in Paris in the International Bureau of Weights and Measures. For PCR efficiency, there are no such standards. You have nothing to compare to.

You can only say how PRECISE is the measurement of efficiency or R2 in your experiment. The only way to do it is to repeat the same experiment many, many times and get standard deviation or another similar statistical analysis that will show you how much variation there is between your repeats.

R2 value determines the phenotypic variance and it should be high where as marker p value should be low in order to determine that marker is a strongest QTL in association mapping experiments. rather than QTL Mapping

e-value above 100% tells you about successful amplification, however with no guarantee regarding specificity, if r2 below 0,998

Hi,

Concerning r-sqared (is my repeatibility good enough? can i measure my sample in this range?):

r-sqared or determination coefficient is just a measure of adjustment of the data points inside a adjusted line that predicts Y(Cq) as a linear function of X (log (DNAcopies)). It ranges from 0 to 1; 1 meaning a perfect inclinated line.

In this particular case, a bad r-sqared means that your standard curve technical replicates are not close each other (low repetibility) or your serial dilution for standard curve are not well done.

Concerning efficiency (is the reaction working as supposed to be? - double each cycle):

The efficiency means how much amplicons are produced after each cycle. If everything is OK, is expected that your product, amplicons, double at each cycle following the exponential equation: Y=Ci.(1+e)cycle. Ci=initial concenration of target; e=efficiency.

If the efficiency is 100% (e=1), the Cq values of the 10 fold dilution will be 3.3 cycles apart (there is a 2-fold change for each change in Cq).

Bad efficiency means that you have problems with pcr inhibitors, primers inespecificity or other problems, including kit expiration date.

Solutions:

A) Solution for a bad r-sqared and good efficiency: try a new standard curve preparation, mix well the tubes. Pipet sample and qPCR mixes in a 40%/60% ratio to avoid subsampling error in the low concentrations points. Example: 8uL sample and 12uL qpcr mix. Calibrate pipets.

B) Solution good r-sqared and bad efficiency: qPCR are very precise between 15-25 cycles, all your points are in this range? Above cycle 30 qPCR are not precise. Use your standard curve between 15-25 cycles, dilute your samples to amplify in this range. See the melting curve and evaluate also running a gel.

Primers specificity tests: Sometimes two peak melt are not two products, but two stage melting curve of a single product, test in silico (https://www.dna.utah.edu/umelt/um.php ). Test your primers for dimers and hairpins in silico ( https://www.idtdna.com/pages) . Confirm you amplicon size (80-200bp). Blast your primers in ncbi just to check.

Best,

Jacó