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Standard curve


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#1 seed

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Posted 31 October 2011 - 07:33 AM

I'm new with RT-PCR. Can someone explain to me how can I make standard curve from my dilution series? What should I dilute, my sample (cDNA) or my housekeeping gene (UBQ)? And how can I make the dilution series?

#2 doxorubicin

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Posted 08 November 2011 - 05:12 PM

Start with the cDNA that you will be using. You need to generate a standard curve for every gene you will be analyzing including the housekeeping gene. In a separate well (or a couple wells) for each primer set, run a PCR like you will be doing when you analyze your samples (same master mix, same machine). When the reaction is done, make sure your melting curves are clean (it's also good to check the molecular weight of the band on an agarose gel and to sequence the product), then purify the product using a Qiaquick kit or equivalent. Check the concentration (nanodrop) of your purified product (this will allow you to know the absolute quantity of product)....and dilute it in a series 1:5 or 1:10 in TE buffer with sheared salmon sperm DNA. Run your standards on your qPCR machine using the primers you used to generate them and determine which part of the dilution series gives you a linear (R-squared near one) result with an Efficiency less than or equal to 100%. These dilutions will then be your standard curve that you will run along side your samples when you asses the quantity of cDNA present.

#3 aj00016

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Posted 20 April 2012 - 02:22 PM

Hi,

I'm having the same issue, as I don't know how to generate my standard curve.

Does that mean you need to
1- Run a normal PCR using primers against your target genes as well as the housekeeping gene, run these on a gel and purify/extract the appropriate bands, find out the concentration using a nano-drop, make serial-dilution in TE buffer, and run these on qPCR machine to get the standard curve?
2-You then run the cDNA from your sample and compare the Ct to the standard curve from step (1) to determine the concentration of the unknown sample?

If the above method is correct, then why would you need a standard curve for house keeping gene, can't you compare the Ct value of target gene from sample with that of the standard curve generated after in step (1)?

Any comments/help will be greatly appreciated :)

#4 doxorubicin

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Posted 15 May 2012 - 01:10 AM

Sorry for the late answer.
1) If by "normal PCR" you mean something other than "same master mix, same machine", then no. Run the PCR precisely as you will be doing when you do the qPCR. Then just peel off the lid of the 96 well plate and collect the finished PCR product to purify it as indicated above. Use someone else's bench and pipettemen, so you don't contaminate yours with PCR product. If you are cutting out bands, then your PCR is not clean enough for qPCR. You will need to redesign your primers. If you have a clean melting curve, purify from solution using Qiaquick, get concentration from nanodrop, send some for sequencing, and make dilution series with the rest.

2) Yes

And finally, to this question: "If the above method is correct, then why would you need a standard curve for house keeping gene, can't you compare the Ct value of target gene from sample with that of the standard curve generated after in step (1)?"

Response: I think you are asking if you can run the standard curve one time and assume that the machine will always give precisely the same values when the same curve is run in the future. In theory, I guess this is correct, but in practice, there will likely be a great deal of variability due to things you can control (pipetting, how long the plate sat at room temperature, etc.) and things you can't control (lot-to-lot variability of master mix, fluctuations in machine performance, etc.). As is often the case, it is probably fine to have one reference standard curve that you work off of, but you can always be more rigorous and do things in a more costly manner to be more precise if you need to be.




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