Over 100% Assay Efficiency in qPCR? Not so fast. -- Ask TaqMan #21
HTML-код
- Опубликовано: 27 фев 2014
- Submit your question: bit.ly/1cgFftk
In another Ask TaqMan video, we discussed assay efficiency and how to calculate it. But how does efficiency really impact our experiments, or as Julie Kase at George Washington University asks, why is it important? Does the efficiency for all of my targets have to be the same? The aim for all assays is to be 100% efficient, which means there is an exact doubling of your template every cycle.
We previously found that this can be calculated thru a dilution series and the following equation. In order to use the ΔΔCT method for relative quantitation, the efficiency of the target and endogenous control must be approximately equal - meaning that the assay efficiency should be within 10% of each other, so 100% +/ 10%. If the range is greater than 10%, then you need to be aware that when you evaluate fold changes, the unequal PCR efficiencies will correlate to a decrease in the accuracy of the calculated fold change.
Keep in mind that Life Technologies has well over one million pre-developed Assays, which have been in-silico validated to be up to 100% efficient, so the efficiency calculation is not necessary. However, If you do check, and find the efficiency is outside of the acceptable range, then there is likely something from the sample or setup that is throwing things off.
So what happens if my efficiency is say, 150%? Your first thought might be, "Wow, my assay rocks! That's good, right?" Well, actually no. It is not possible to be over 100% efficient, so something else is going on here. Let's review some causes of high or low efficiency, and how to fix them.
First, let's take a look at what's going on with efficiency over 100%. The most common cause of efficiencies greater than 100% are inhibitors. This can be carryover from the sample itself such as heparin or humic acids. Or another source could be contaminants from the RNA or DNA isolation such as SDS or phenol. 
Наука
Or simply, if efficiency is over 110%, use a more diluted cDNA sample for your assay. as long as your gene does not rise very late (Ct 32-35), use a 2-10 fold dilution of cDNA in your assay, which will also further dilute the PCR inhibitors as well and will ensure an efficient PCR replication
In my case, Beta actin slope is -2.35 (maybe efficiency 130~150%). Ct level is 26~28 and melting point is 89.7(it's constant in every experiment). RNA 260/230 levels are 1.68~1.95, 260/280 levels are 1.66~1.71. Is there a lot of inhibitor(phenol, ethanol) in my RNA?.... What's wrong with my experiment? (sorry, my english level is very poor. but I'm so frustrated in experiment. Help me please..!)
Hi there. Thank you for the video. I wonder if there is any reference I can cite for PCR efficiency over 100%.
Hi Marty. Thanks for your question. We have information on real-time PCR efficiency and I have linked to it below.
If you do have any additional technical questions, please contact us at thermofisher.com/askaquestion. Thank you!
www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/real-time-pcr-basics/efficiency-real-time-pcr-qpcr.html#bulletin
hi
i made an electromotor with 133% efficiency
4000rpm . 2kw out put
in put 48 v . 1500 watt
if you like i can share my experimental datasheet
I miss Doug :(
what is the difference b/n dCt and Ct value? thank you i love the life technologies
Hi Yenesew, please contact our technical support team at thermofisher.com/askaquestion for additional information. Thank you!
Does it good to have 112.5% of effeciency?
Thank you for your question. We recommend having an efficiency between 90 and 110%. Reactions that have an efficiency over 110% may be due to poor RNA/DNA quality, having contaminants from nucleic acid purification, as well as having a high template concentration. As you’re very close to our recommended efficiency, you can try omitting a data point from either the most concentrated or least concentrated sample to see if your efficiency improves.
For additional technical support, please contact us at Thermofisher.com/askaquestion. Thank you.
Where is Doug?
We wanted to change it up a little and feature some other very talented experts within Life Technologies.
Could she then be Taqwoman?
Can you describe DNA sequencing in detail! Please not the sanger-termination method. Something more modern would be nice!
Sure thing! Ion Torrent, one of the Life Technologies brands, utilizes technology called "Semiconductor Sequencing". Semiconductor sequencing uses a combination of chemistry and semiconductors to sequence DNA using, what is essentially the world's smallest pH meter to determine what bases (T, C, A, G) are called based on the change in hydrogen ions (H+) in solution, as described in this technology overview video on the Ion Torrent Sequencing Technology webpage: www.lifetechnologies.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-technology.html.
Bring back Doug!