1) make a dilution series of a standard (it is not important what dilution factors you use; you must only know the factors used. It is most simple to create serial dilutions with a constant factor between subsequent dilutions, e.g. a 1:10 or a 1:2 dilution series). To get a first impression, I would recommend to start with a 1:10 series with 10-15 dilution steps. After having the results from this experiment you will be able to define a resonable concentration range range and go for higher resulution series (1:2 for instance) to record the actual series.
2) plot the ct-values against the logarithm of the dilution factors (DF). In the useable concentration range there should be a linear relationship between dt and log(DF).
3) the standard curve is the regression line through these points. You can use the regression line to determine the concentration of unknown samples from their ct-values, *relative* to the standard (if you know the concentration of your standard, you can calculate the concentration of the unknown samples).
3b) The slope ("m") of the regression line is related to the amplification efficiency ("e") by e = b^(-1/m) where "b" is the base of the logarithm you used in step 2. The value of e should be close to 2. When it is higher you may have problems with background correction in any other part of the data processing. When it is lower, the PCR conditions may be suboptimal (presence of inhibitors...). A value close to 2 does not exclude the possibility of such problems, but at least it gives no clear indication that problems do exist.
4) Good luck.
1) make a dilution series of a standard (it is not important what dilution factors you use; you must only know the factors used. It is most simple to create serial dilutions with a constant factor between subsequent dilutions, e.g. a 1:10 or a 1:2 dilution series). To get a first impression, I would recommend to start with a 1:10 series with 10-15 dilution steps. After having the results from this experiment you will be able to define a resonable concentration range range and go for higher resulution series (1:2 for instance) to record the actual series.
2) plot the ct-values against the logarithm of the dilution factors (DF). In the useable concentration range there should be a linear relationship between dt and log(DF).
3) the standard curve is the regression line through these points. You can use the regression line to determine the concentration of unknown samples from their ct-values, *relative* to the standard (if you know the concentration of your standard, you can calculate the concentration of the unknown samples).
3b) The slope ("m") of the regression line is related to the amplification efficiency ("e") by e = b^(-1/m) where "b" is the base of the logarithm you used in step 2. The value of e should be close to 2. When it is higher you may have problems with background correction in any other part of the data processing. When it is lower, the PCR conditions may be suboptimal (presence of inhibitors...). A value close to 2 does not exclude the possibility of such problems, but at least it gives no clear indication that problems do exist.
4) Good luck.
Mr.Wilhelm is correct. A simple serial dilution will give you a good standard curve by which to compare your experimental values against. I always made my standards with a known DNA sample and actin primers (or something else highly conserved).
Which cycler do you use? The most cyclers have special tools for standard curves..
I think this forum is not a place for learning methods.There are plenty of resourses in the web, and there are many good books about real-time PCR. Also the Lightcycler (as well as all other instruments) come with user manuals in which these thing are explained. You should first read and learn something about the technology, method, and analysis. After this, you can ask specific questions that can be successfully answered in such a forum.
There is a good guide by Bio-Rad for beginning qPCR. You can find it on web. Otherwise I can send it to you..It is very useful for anyone, start up with qPCR. It also concludes explanation about standard curves.
To plot standard curve for primers, do I always need positive samples which express my gene of interest? I am wondering if it can work for unknown samples too.
A standard should have a relatively high concentration of the target sequence, so that you can create a dilution series over many dilution steps.
Using a cDNA right away as standard is usually a bad idea.
Prepare a standard from a PCR, purify the PCR product, determine the concentration, dilute it to a good working concentration and use this as your standard (to prepare dilution series). NOTE: prepare the standard in a different lab! Take only the working concentration back to the lab where you set up the qPCRs.
It is often a good idea to use "outer" primers to generate the standard, so that the standard is a PCR product longer than the product produces in the qPCR that contains the qPCR primer binding sites.
To prepare a standard with PCR you will initially need some template, for sure. The obvious source is cDNA from a cell expressing the gene. If this is absolutely not available, you may order a synthetic DNA fragment with the target sequence that you can amplify by PCR.
Dear Panagiotis Apostolou,
could you please send me the guide by Bio-Rad, because I have the same problem in creating the standard curve.
thank you
I know this post is a bit old but I found I wanted to share this link which I found useful since I couldn't get high concentrations from my gel extractions. http://www6.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf
Please help me to make a serial dilution for standard curve in real time PCR .I have the DNA sample of cancerous patient that OD is 510 ng/μl. In my hand there is no other information about this sample.
A human genome has 3,234.83 Mb, a base pair has an average molecular weight of 660 g/mol.
A standard should have a relatively high concentration of the target sequence, so that you can create a dilution series over many dilution steps.
Using a cDNA right away as standard is usually a bad idea.
Jochen Wilhelm
Hello, I did all the steps you've mentioned and I have a question about this part of what you've said:
"dilute it to a good working concentration and use this as your standard (to prepare dilution series)"
I was told to do a 10-fold dilution starting with 10 million copies and, just as an example, in one of the templates I have 27.5 × 10^9 Copies/μl.
I am beginner in qPCR and I couldn't figure out how to do the calculation in this part. The purified PCR products that I have now are ~ 26μl. I don't know how much I should use. (totally confused!)
I will appreciate your help.
Firstly, I would recommend doing a 2-fold dilution series rather than a 10-fold.
Starting with 10 million = 107 copies will give you a lowest Ct value of somewhere around 14, what is ok. You can do 24 1:2 dilution steps. The highest dilution will then have an expected number of 1.2 copies.
If you add 2 µl per PCR reaction, and the undiluted standard should contain 107 copies, you have to perpare the standard to have a concentration of 107 copies/2µl = 5 × 106 copies/µl.
Your question is how to dilute 27.5 × 109 copies/µl down to 5 × 106 copies/µl?
There are many ways to achieve this. I would do it in several steps. Let's call your purified PCR product with 27.5 × 109 copies/µl solution A.
1) dilute A to 5 × 109 copies/µl (5.58-fold: 10 µl A + 45.8 µl water) --> B
2) dilute B to 5 × 108 copies/µl (10: 10 µl B + 90 µl water) --> C
3) dilute C to 5 × 107 copies/µl (10: 10 µl C + 90 µl water) --> D
4) dilute D to 5 × 106 copies/µl (10: 10 µl D + 90 µl water) --> E
Here we are. Solution E is the standard you use. If you add 2µl of E to a PCR, it will contain 107 copies. A, B, C and D will stay in the "post PCR lab". You should take ONLY E to the place/lab where you are going to prepare the PCRs. Change gloves and lab coat.
If you prepare the 1:2 dilution series, I recommend to prepare a series of 23 reaction tubes, each containing 5 µl water. Then take the very same pipet (don't change anything; you don't even need to take a new tip!), take 5 µl of E and mix it to the 1st 5 µl water. Then take 5 µl of this and carry it over to the next and so on. You will end up with about 95 µl E, 5 µl of the fistst 23 dilutions and 10 µl of the final dilution.
Although I think that single reactions in a series with 24 concentrations is sufficient to get a reliable result, the volume is enough to make duplicates (if you want and if space and money is not limitting).
Jochen Wilhelm Thank you so much for your time and detailed answer.
I didn't understand the part " (5.58-fold: 10 µl A + 45.8 µl water) --> B".
Also, when they say a dilution series starting with 10 million copies, shouldn't be 10 millions down to 1000 copies? (if we use each diluted standards (B,C,D,..) duplicate in a qpcr plate)
A,B,C,D are just steps to create your standrad. E is your standard. You are NOT!!! using A,B,C,D in your PCR!! They are much too concentrated.
@Jochen Wilhelm ,Is all the 24 dilution series are required for the construction of standard curve? And, 1 in 2 dilutions is okay rather than 1 in 10 dilutions?
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