I need to do some qPCR on qDNA samples that are at very low concentrations, such as 0.5-1 ng/ul. I have very few samples left, and I was wondering if anyone knows the minimum amount of DNA that can be detected in a qPCR (SYBR mix) reaction. I know it will depend on manufacturer of the Master Mix used. Thank you in advance.
Hi Catarina,
Our lab work with virus. We could detect virus DNA copy number with SYBR Green. As far as experiment goes. If you have a good primer design and your qDNA is processed to get rid of contamination you can detect 1pM on an amplicon that is 100-200bp in length. That is roughly 10 copies of DNA/uL. What you could do is generate a dilution standard by normal PCR. Run on agarose gel with a standard ladder to figure out the concentration. Calculate the copies number on your standard. Do a test run of your standard curve with a diluted standard roughly equal to your sample concentration. In that way, you don't waste your sample and at the same time test out the bottom threshold of your detection detection assay.
Good luck.
Hi Catarina,
Our lab work with virus. We could detect virus DNA copy number with SYBR Green. As far as experiment goes. If you have a good primer design and your qDNA is processed to get rid of contamination you can detect 1pM on an amplicon that is 100-200bp in length. That is roughly 10 copies of DNA/uL. What you could do is generate a dilution standard by normal PCR. Run on agarose gel with a standard ladder to figure out the concentration. Calculate the copies number on your standard. Do a test run of your standard curve with a diluted standard roughly equal to your sample concentration. In that way, you don't waste your sample and at the same time test out the bottom threshold of your detection detection assay.
Good luck.
I think that 0.5-1 ng/ul is a fair DNA concentration. A minimum 10-15 pg DNA/reaction (40 cycles) may be enough (actually it is enough), since 1 human cell contains is roughly 7 pg of DNA. The problem when such low quantities of DNA is used is the poor reproducibility, because of the stochastic effect on sampling. I think the optimal DNA cc. range in a 20-25 uL volume is 0.1-50 ng/reaction. Diluting your sample to 0.05-0.1 pg/ul can be OK. If you are going to perform reliable quantitation, keep in mind that the concentration of the most diluted sample should be higher than concentration of the second lowest calibrator. Also check the non template control amplification, since with sybrgreen chemistry primer dimers may give false 'amplification' even without DNA.
Bottom line: check your qPCR sensitivity with a standard curve in a 5 (3?)x dilution steps down to 10-15 pg DNA/reaction, check the NTC amplification, and you will know that diluting to 0.05-0.1 pg/ul will work or not.
Have luck!
I agree with all the above answers and to add some more to it, make sure your qDNA is endotoxin free. In the presence of endotoxin, low concentrations of DNA are poorly amplified.
Hi all, thank you very much for your replies, I'll follow your suggestions, fingers crossed!
Hi Catarina,
I do not know if you use human DNA, but in case of human DNA, 1pg (picogram) contains 1 copy of a single gene. Thus, if you have 1 ng/ul you have enough DNA to do your qPCR.
Ioannis
Sorry, I make a mistake. 3,3 pg (picogram) of human DNA contain 1 copy of a single gene. This is based on the estimation of 3,4e9 bp human haploid genome.
Ioannis
Hello Catarina, it will always depend on the sample purity, the Taq polymerase you are using and the type of qPCR to follow. Theoretically, if your tube contains a DNA molecule, the Taq should detect and amplify it. However, many researchers and companies state that the theoretical limit of detection (LOD) by qPCR at 95% confidence is of 3 molecules. Using genomic DNA or cDNA also will affect the minimum concentration required. You will also find differences between species, where in 1pg you could have represented different numbers of copies for a single gene. The ratio for "weight/number of copies" in plants may vary much more cause polyploidy. I suggest to begin using ≥ 10 pg DNA (you can have a dynamic range from 100ng to 10pg). Best luck!
Hi, a masters student in our lab also had very low concentrations for their pRT-PCRs and they found a protocol that requires the addition of tRNA to the reaction and this helped them get better amplification and and more consistent results...you could try search for this technique. I unfortunately don't have the protocol for it.
Usually tRNA is used in the extraction as carrier... , maybe it can enhance the low quanity DNA sampling as well
Hello again, thank you all for your helpful replies. Would yo suggest any specific MasterMix to be used? The one we use at the moment requires a minimum of 2ng per well (which is too much considering my samples), do you know if there are mixes specifically designed for low amount template? Thank you once again. Best wishes.
Hi Ayodele, yes, I don't know of any organism in which it cannot be done, as long as the primers are specific and efficient, and the gDNA/cDNA is of good quality.
Thanks i appreciate. The primer i intend to used is specific for acetylcholinesterase gene
go ahead with pico gram concenrations, it can even do femtograms by ur priomers should be best designed to aplyfy.. no primer dimers..
Hello people. I'm having the same problem as Catarina. In fact, I have less than 0.5 ng per microliter, becouse I work with very small parasite. And I can not amplify any pcr product. can you help me? could you give me some suggestion of mix that could help? thanks a lot
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