Hi,
I would like to know the cDNA quantity you recommend for the mentioned PCR. It will be used to validate RNA-Seq results.
Thanks, Bernardo
Hi Bernardo,
It depends on your cDNA quality, target gene and PCR efficiency which you can find out it by serial dilution test. We performed some experiments with 1 ng of cDNA per reaction.
I recommend you to prepare serial dilution base on cDNA concentration to find the optimal concentration to have higher PCR efficiency. Since you want to use ΔCt method, PCR efficiency is so important.
As much as required to have a minimum of about 100 target molecules per reaction (below this amount the Poisson distribution of molecules makes a precise quantification difficult).
Hi Bernardo,
It depends on your cDNA quality, target gene and PCR efficiency which you can find out it by serial dilution test. We performed some experiments with 1 ng of cDNA per reaction.
I recommend you to prepare serial dilution base on cDNA concentration to find the optimal concentration to have higher PCR efficiency. Since you want to use ΔCt method, PCR efficiency is so important.
More is not necessarily better. With PCR, large amounts of nucleic acid can sometimes gunk up the reaction, so yes -- as the last person suggested. Try a few concentrations until you are in the middle of your standard. It comes a little with experience with your system. I tend to think that the lower quantitative limit is more like a 1000 copies, though 100 is frequently used as suggested by JW.
For a 20 uL RT-qPCR using the ΔCt-method I have used up to 2ug of RNA, but it really depends on how well your gene of interest is expressed. As Somayeh Jamali suggested, it may be worthwhile to perform a serial dillution to test which amount is best.
8ng cDNA is usually sufficient for highly expressed genes, like housekeeping genes (we use GAPDH and 18s). A serial dilution test would put you in the correct range.
Hi, Bernardo
How much cDNA was used in the PCR depend on PCR reaction system, the expression of your genes and the brands of the reagent. your should try a serial dilution test to find the correct quantity.
Another thing you should pay attention. When you conduct the reverse transcription, the same quantity of total RNA should be used in all reaction. In our study, 400ng total RNA was used in 10ul reaction system using takara RT reagent kit.
Hi Bernardo
To all what was said before, I would add that, it is good to work with dillutionsof cDNA which give the Ct values between 25 and 30, and obviously keep the same quantity for testing all the genes.
Hi all,
If I have a concentration of 15ug/mL, so I should do serial dilution, with 15ug/mL as the biggest concentration, right? ANd other concentration would be, 7.5ug/ml, 3.75ug/ml, 1.875ug/ml and 0.9375ug/ml? Thank you in advance.
Hey Jochen Wilhelm and Boris Wawrik
Is there any publication on the minimum "number" i.e. read counts of RNA that can be detected with RT-PCR that you know of?
Thanks!
If a single target molecule is in the PCR, it is amplified. Since you can amplify it to any (detectable) extent, the detection limit is a single molecule.
However, it is difficult to ensure that you have a single target molecule in your PCR. If you use dilutions, you get a Poisson distribution, and -given all your initial measurements and dilutions were correct- the final dilution that has an expected concentration of one copy will sometimes happen to have have no copy, sometimes one, and sometimes two or even more copies (according to the Poisson distribution with expectation 1 you will have about 37% of PCRs not containing any copy, 37% containing exactly one copy and 26% containing two or more copies).
If you talk about RT-PCR (and how many RNA molecules you might be able to detect), the problem with the Poisson distribution doubles, as you might have 0, 1, 2, ... RNA molecules in the RT reaction and from that get again 0, 1, 2, ... cDNA copies into your PCR.
There are few papers known to me that really get this on the point. However, there are many papers more or less indirectly demontrating that this is in fact the theoretical detection limit, like
https://www.nature.com/articles/srep34554
Article Methods to determine limit of detection and limit of quantif...
see also:
http://www.douglasscientific.com/Content/pdfs/AppNotes/IQAPP-PCR-Sensitivity.pdf
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