Scientist Solutions: International Life Science Community By Scientists For Scientists
    

Thanks to our sponsors who make this site possible

qPCR efficiency

RSS Feed

Would you like to save this topic, event, protocol or job so you can find it again easily?

Just click the "Save to My Lab Drawer" link and the item will be saved in the My Lab Drawer section of your bench space.

Available to members only. Please log in or register for your free account now.

danushy
Argentina

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Topic Started by danushy
on 5/13/2009 10:16 AM   
Reply to this post Go to the top of the page

Hi, I am running a qPCR using absolute calibration to calculate copy number of my gene of interest. I have problems with the efficiency of the reaction. The r2 is great 0.999, so that part I assume is OK. But the efficiency is 70 or 80 %. I checked the primers, the annealing temperature is ok, in the melting curve I see only one pick, and the non template has a very small one, but when I run a gel, there is no product. So I am asking what can I do to improve my efficiency. Thanks.


Replies
Ivan
United States

Send PM
See Mini bio

Status: DNA Moderator
Frog Laureate
Posted By Ivan
on 5/13/2009 10:56 AM   
Reply to this post Go to the top of the page

 


Hi danushy,


Low efficiency is typically a result of primer dimers or an assay that has not been properly optimized. It is also always possible that the assay was not designed properly. Do all the dilutions that make up your standard curve yield nice sigmoidal amplification curves? All the R2 tells you is that your pipetting technique is good so it says little about how well the assay is behaving. Could you provide more information about how you calculated your efficiency? What is the slope of your curve? I assume it is steeper than -3.8. Also, how did you set up your dilutions? Did you run 10-fold dilutions and prepared at least 5 different dilutions for your standard curve?


 

Ivan Delgado Orlic
Carlsbad, CA



danushy
Argentina

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Posted By danushy
on 5/13/2009 11:10 AM   
Reply to this post Go to the top of the page

Hi!!! Thank you for your answer.
All the points of the standard curve have nice sigmoidal amplification curves, the slope is sometimes arround -4 and now is -3.874. So now it is better. But the efficiency is 81%.
As I told you, I checked the annealing temperature, twice.
The efficiency is calculated with the iCycler program when I do the PCR standard curve, I obtain the slope, the efficiency and the correlation coefficient. The only thing I observe is in the melting curve the non template has a very small pick arround 78 , andt the Tm of the product is 81.5. Could this be the problem? But when I run an agarose gel, there is no band!!! So I don′t undestand what is going on.
I do a 10-fold dilutions with 7 different dilutions. (100 pg, 50 pg, 10 pg, 1 pg, 0.1 pg, 0.01pg, 0.001pg)
Thank you for your help!!!!



Ivan
United States

Send PM
See Mini bio

Status: DNA Moderator
Frog Laureate
Posted By Ivan
on 5/13/2009 11:41 AM   
Reply to this post Go to the top of the page

 


 


I think I have a better answer for your problem now. First of all, I strongly suggest you prepare a different set of dilutions (and I would not go lower than 10 pg; 1 pg if you must). I do not know what your DNA source is, but 0.001 pg is a very small amount of DNA. Considering that a single cell has 6 pg of DNA, and your target is a single copy gene, you cannot detect 0.001 pg of DNA. Along these lines, what Ct value do you obtain for your 0.001 pg dilution? If it is higher than Ct 35 then you are pushing your assay a little too much. 


I would not worry too much about the tiny peak in your negative control. As long as the signal is minimal, and the Ct of your negative control is significantly higher than the highest Ct on your standard curve, you should be fine. qPCR is a thousand fold more sensitive than an agarose gel so not being able to see a band in a gel does not really say much about how dirty your qPCR assay is. 


Another thing to consider, if you designed an assay against a sequence that has multiple copies in the genome you are studying, is that you may be amplifying more than one sequence at the same time (which may not be distinguishable in your melt curves). If this is the case, and your DNA template has a particular DNA sequence, you may end up with weird results like these. Unfortunately if that is the case, you may need to redesign your assay. This is unlikely, but possible.


 

Ivan Delgado Orlic
Carlsbad, CA



mithu_du_bmb
Bangladesh

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Posted By mithu_du_bmb
on 7/10/2009 9:04 AM   
Reply to this post Go to the top of the page

Would you please check your product length of RT-PCR? Usually small DNA fragment is difficult to see in gel run.



RomeoLima
Canada

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Posted By RomeoLima
on 5/25/2010 20:51 PM   
Reply to this post Go to the top of the page

Have you tried constructing the standard curve just using  five points instead of seven? Selecting all the points can give you a good R^2, but at the same time if one of the points is reading beyond the actual limit of detection of your qPCR, then your efficiency can be as low as 70%. To make another set of standards is a really good idea!!!. I would suggest you to use instead of ten fold, a two or a five fold serial dilution.

 

The small peak that appear in your qPCR data could be due to primers dimers, since the melting temperature is lower than the corresponding to your target. If there is no Cq (or Cp) in your NTC, then do not worry to much  about that small peak!!



Good Luck

 

! - )



RL



As a Scientist Solutions member, you are able to register a positive vote for any topic which you believe is useful and relevant to our board or any reply which you believe is especially well worded and helpful.

By participating in the voting, you will be helping to identify the best topics & replies on the board.

You may vote once for any one post, and you may not vote for your own posts.

A post (topic or reply) will earn one "thumbs up" icon for every 10 votes received (up to 3 thumbs up), and the person who made the post will also earn two bonus points.

learn more about member points.