We are using TaqMan Advanced kit, the efficiency in most of our standard curves is 150%, with low slopes (-2,6) but high R2 (>0.99), how can I improve results?
Has changing the dilutions (currently using 1:10, 5 folds of 1 ng starting tissue sample), narrowing the dinamic range or other solution worked for you?
And until what values can I accept my results as reliable?
Thanks!
Camilla,
In my opinion, you should perform 2-fold dilutions of your standard control until you reach linearity. After this is achieved, you can "spike" dilutions of your unknown to see how it interferes with the standard. Search for a discussion of MIQE as a guideline of approaches to solving your query.
Let the board know of your success
Michael
Hi Camila
Usually a high efficiency occurs due to the presence of an inhibitor. How are you purifying your miRNA? Are you using a kit? How are your 260/280 and 260/230 ratios? You can use lower concentrations of your standard curve (that will dilute the inhibitor alowing a linear curve). Another possíbility is try using a kit, which I recommend for miRNA, miRVana is very good.
As we know, 150% qPCR standard curve efficiency is highly problematic. Without knowing what you are using for your standards, it seems possible that your first 1 or 2 points on your present standard curves are inhibited (too much sample and any unidentified inhibitory carry-over material in the reactions; giving Cq values that are artificially too high - on account of inhibition). Eliminate the first point and recalculate your efficiency(s) - what do you find? Then eliminate the first 2 points and recalculate - what do you find? Answers to these things would help.
On another angle: your high correlation (R2 values) at present could indicate that your probe has high background - either pre-cleaved already and/or not purified of free fluor from the outset upon purchase. This could cause a stable flat-lining of your curve at all points and end up exaggerating your observed efficiency(s) above 100%. This 'flat-lining' is usually observed with SYBR Green-based qPCR plagued by primer dimer and/or non-specific product formations at the later points in a standard curve...
Agree with Michael that I only do 1:3 dilutions and find that companies' claims of many orders of magnitude linearity almost never work in practice. It would be helpful to know what Cp's you're looking at. I find for most primer sets between 18 and 27 to be the sweet spot for linearity. In answer to your last question, we usually consider a slope between -3.0 and -3.6 to be good enough. You can correct for the difference in efficiency using the Pfaffl method.
http://www.ncbi.nlm.nih.gov/pubmed/11328886
So you mean 1 ng of cDNA or RNA you're loading? Are you doing the RT in the well or separately? Have you tried the spike in controls?
Thank you everybody for your answers.
Andre: We used miRVana kit for total RNA isolation from mice myocardium. We tested RNA quality with 2100 Bioanalyzer Agilent (RIN=8 approx).
Michael and Jacob: We are currently using 1:5 dilutions, so we will consider making new experiments with lower dilutions, because our ΔCt ranges are: 25-33. Our slopes have values around -3.1 and -2.7
Jack: We perfomed cDNA Synthesis with TaqMan™ Advanced miRNA cDNA Synthesis protocol.
We have analized the amplification curve of our samples. We realized that the final flourescence of high concentration (1:10) dilutions is lower than the low concentration (1:100) dilutions (see picture attached). We think that the presence of inhibitors in the high concentration dilutions reduce pcr reaction, what do you think?
Thank you again for your kindly advice.
I agree your data suggests inhibitors. Also consider that high Cts can sometimes indicate non-specific amplification. Have you run the products on a gel? Are you able to purify a band and send it for sequencing?
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA