Very low efficiency of qPCR

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huamei
huamei's picture
Very low efficiency of qPCR

Dear Sir/Madame,
I have low efficiency problem with qPCR. My primers were designed based on the varible regions of 16S rRNA gene as I want to know the exact copy numbers of 16S rRNA gene. My standard curve was made from plasmid inserted with my target. The target fragment was cloned using topo TA cloning kit for sequecing. The plasmid was extracted and digested by restriction enzyme Pst I and the band was cutted from gel. I have changed thermal protocol and changed another pair of primers. The efficiency is still 60%-70%. The reaction components per 20 μL were 7 μL H2O, 10 μL 2x Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies) and 1 μL 10 μM of each primer.

I just hope whether your science support could help me figure out the problem, as you are the expert of qPCR.
Many thanks and look forward to your reply.
Best regards!
 

Ivan Delgado
Ivan Delgado's picture
Hi huamei,

Hi huamei,

The first thought that comes to mind about your problem is the DNA fragment you've purified to use as template. Is this fragment large enough to have at least 100 bp of DNA flanking the region your primers amplify? Taq polymerase needs at least a little DNA to grab hold of the template before it starts extending the primers, so unless your template is larger than the region you are amplifying the PCR is not going to work very well. 

Another consideration, given that you are going through a number of steps to prepare your DNA template, is the quality and concentration of your DNA template. How much DNA are you using per PCR reaction? If your DNA is not concentrated enough, or somewhat degraded, you are not going to get good PCR amplification. 

Hope this helps. My recommendation is to use genomic DNA if at all possible to optimize your qPCR. Once you know the assay is working, then test it using the purified DNA template you describe. 

huamei
huamei's picture
Thank you for your reply,

Thank you for your reply, Ivan.

My DNA fragement is about 185bp. The standard curve was generated using triplicate 5-fold dilutions of plasmid DNA from 3e+006 to 9.6e+002 copy numbers. I think that the length of DNA fragment and template concentration are fine. No detectable peaks that were associated with primer dimer were observed in melting curves.But the melt-curve peaks showed small differences (less than 1 °c). Is this a problem?

And another mybe stupid question, after I isolated the plasmid inserted with my target, do I need to linearize the plasmid? I did so, but the result was no difference. I am so upset now. I know there must be something wrong in my qPCR experiment but I tried many times and still can not find it.

All the best

huamei
huamei's picture
Sorry, I have to modify what

Sorry, I have to modify what I posted. There was only one peak in melting curve for one template. But for different dilutions, the peaks had small difference (less than 1 degree).

Ivan Delgado
Ivan Delgado's picture
Hi Humei,

Hi Humei,

I am not sure if you realize that your response does not answer my questions in any way. Saying that "I think that the length of the DNA fragment and template concentration are fine" really means nothing. I do not know if you truly understand if the DNA fragment size is fine, or if the concentration of the DNA is fine, unless you share that information here. 

If your DNA fragment is 185 bp as you pointed out, and you are amplifying a 180 bp fragment with your primers, then that is the reason why your PCR is not working: the amplicon is too similar in size to the DNA fragment you've purified, which gives almost to space for the polymerase to grab the DNA during amplification. 

If your DNA concetration is below 10 nanograms per reaction then that too could explain why your PCR is not working. Purifying a DNA fragment the way you do is not very efficient and you tend to lose a lot of that DNA in the process, so it is quite possible you simply do not have enough DNA for the PCR to work well. 

Having melting peaks that are 1oC different can be a problem. Such peak shifts are actually quite significant. This could be due to the reasons I explained above.

This kind of experiments do require that the plasmid be linearized. But as I said in my previous post it would be to your advantage to use high quality genomic DNA to optimize the qPCR first before trying to use your purified plasmid DNA and see if the qPCR works. 

handsomejohanna
handsomejohanna's picture
Parameters that affect the

Parameters that affect the efficiency of PCR

Several parameters can affect the efficiency of the PCR (ranked from most to least frequent, based on our observations at Applied Biosystems Support):

  • Your samples may contain PCR inhibitors
  • Your PCR primer and/or probe design may not be optimal
  • Inaccurate sample and reagent pipetting
  • The standard curve may not have been properly analyzed
huamei
huamei's picture
Thank you very much, Ivan and

Thank you very much, Ivan and Johanna.

Yes, I think my DNA template concentration is too low (less than 10ng per reacion). This probably is the problem. The size of my plasmid template is 3956bp and my amplicon is about 180bp.

I will try again according to your advice. Hope it will works this time.

Thank you again.

huamei
huamei's picture
Sorry, after I asked a

Sorry, after I asked a colleague in my lab, I am confused again. My DNA concentration is 15.8ng/ul and the DNA fragment (linearized plasmid inserted with my target) is 4208bp. So, assuming that the average molecular mass of a double-stranded DNA molecule is 660 g/mol, the DNA copies is 3.426e+09 copies/ul. If I make a series of dilutions from this concentration, the copy numbers look fine, but the DNA concentration will be lower and lower. Obviously it will be lower than 10ng. Do I need to care this concentration? Or it is OK if the copy numbers are enough.

Sorry for my questions. But I really want to figure this out.

All the best!

Ivan Delgado
Ivan Delgado's picture
Hi huamei,

Hi huamei,

A concentration of 15.8ng/ul for a 4,208 bp DNA fragment is indeed over 3 billion copies per microliter (I got 3.48e+09). In other words it should be more than enough DNA for your qPCR to work. 

Having said that, it is very important to measure the DNA concentration of your plasmid accurately. Have you measured the DNA concentration using a UV spectrophotometer? If so, did you use DNA standards of known concentration to make sure the UV spec is working correctly? Did you run this plasmid DNA on a gel, after purification, to make sure you have the amount of DNA your UV spec says you have?

It should be perfectly fine to have DNA concentrations below 10 ng when your DNA template is only 4,208 bp long. What is important is the number of templates you have, and you have plenty. A decent qPCR assay should easily detect 100 copies of your template, but as I said above you must be 100% sure that the concentration you believe you have you truly  have. If you really have a plasmid at a 15.8 ng/ul concentration, running a few microliters of this plasmid on an agarose gel should give you a very bright band. 

huamei
huamei's picture
Hi Ivan,

Hi Ivan,

We used Qubit-fluorometer (Invitrogen, Carlsbad, CA, USA) to measure the DNA concentration. Now I wonder if the concentration of my primers are too high (500nM in reaction). But there is no primer-dimer peak in dissociation curve. Do I need to optimize primers concentration from 50-300nM? And the annealing temperatures I tried are from 59-62 degrees, as when I performed tradition PCR at 62 annealing temperature, I could get a single band (showing the primers are quite specific at this temperature). Due to the low efficiency, do I need to try lower annealing temperature?

Many thanks!

Ivan Delgado
Ivan Delgado's picture
Hi huamei,

Hi huamei,

Using a Qubit to measure DNA concentration is fine, but it is also important to determine the DNA quality. To do that you need to measure the 260/280 ratio and also run the DNA on a gel to see that it is not degraded. 

As for primer concentration 500nM is fine. Still, optimizing primer concentration will likely help. I would go as low as 100nM and as high as 1000nM. 

Annealing temperature is definitely an important component that you need to optimize. I would go as low as 56oC and as high as 64oC. If you have a gradient PCR cycler this should be easy to do.

Good luck! 

Curious George
Curious George's picture
With 60-70 % efficiency your

With 60-70 % efficiency your assay is not working and you cannot trust the data. Being in Denmark I suggest you contact TATAA Biocenter and have them design new assay for you.or, if you preferdoing it yourself, join their primer design class.

Good luck!