urgent help-how to get amplification from very low target copy number and how to - "Taqman probe" qPCR and "rotor gene 6000" machine (Oct/03/2007 )
Dear all,
I am newly registered in the forum. I got lots of valuable information from the forum about real time PCR.
But now I am facing big problem about my experiment. I am doing the absolute qPCR with Taqman probe to detect the virus(a kind of DNA virus) copy number in shrimp tissue by Rotor Gene 6000 machine.I want to get a standard curve with a range from 5.9*10^5 to 5.9*10^0 copies/ul with Takara premix(RR039). But I can't get any amplification from 5.9*10^0(I add 5-7.5ul plasmid in 20 ul volume, that means there are about 44 copies per reaction). I use Taq man probe( final conc. is 0.125uM), primer(0.25uM/ul each) and 5-7.5ul plasmid in 20 ul volume. Predenature at 95 degree with 30 sec, then 95 degree denature with 5 sec, annealing and extention at 60 degree with 20-30sec., 40-45cycles(followed Takara protocol). I tried to adjust the ratio of primer and probe, the denature and annealing time, but no improvement. The Ct value of 5.9*10^1 copies plasmid was about 36, but the Ct value of some of my unknown samples were 37-39. Why my palsmid of 5.9*10^0 copies/ul can't get amplified at about 39-40 Ct value(in theoretical, it should be!) but my unknown samples can(100ng gDNA/reaction)? what's the limit copy number can be detected by real time PCR? I optimized the ratio of primer and probe. I also tried 5.9*10^0 copies/ul with different ratio of primer and probe but no improvement. but most of my unknown samples got amplification at 36-39 cycles, that means they contains much lower target copies....
Because in most of my unknown samples there is very little target gene, so I have to get a standard curve including the point of 5.9*10^0. Any suggestion how to do? Is the Ct value higher than 35 reliable? If it's not reliable, how to deal with my unknown samples with higher Ct Value(more than 35)?
I need help very much!Look forward to your answer.
I am newly registered in the forum. I got lots of valuable information from the forum about real time PCR.
But now I am facing big problem about my experiment. I am doing the absolute qPCR with Taqman probe to detect the virus(a kind of DNA virus) copy number in shrimp tissue by Rotor Gene 6000 machine.I want to get a standard curve with a range from 5.9*10^5 to 5.9*10^0 copies/ul with Takara premix(RR039). But I can't get any amplification from 5.9*10^0(I add 5-7.5ul plasmid in 20 ul volume, that means there are about 44 copies per reaction). I use Taq man probe( final conc. is 0.125uM), primer(0.25uM/ul each) and 5-7.5ul plasmid in 20 ul volume. Predenature at 95 degree with 30 sec, then 95 degree denature with 5 sec, annealing and extention at 60 degree with 20-30sec., 40-45cycles(followed Takara protocol). I tried to adjust the ratio of primer and probe, the denature and annealing time, but no improvement. The Ct value of 5.9*10^1 copies plasmid was about 36, but the Ct value of some of my unknown samples were 37-39. Why my palsmid of 5.9*10^0 copies/ul can't get amplified at about 39-40 Ct value(in theoretical, it should be!) but my unknown samples can(100ng gDNA/reaction)? what's the limit copy number can be detected by real time PCR? I optimized the ratio of primer and probe. I also tried 5.9*10^0 copies/ul with different ratio of primer and probe but no improvement. but most of my unknown samples got amplification at 36-39 cycles, that means they contains much lower target copies....
Because in most of my unknown samples there is very little target gene, so I have to get a standard curve including the point of 5.9*10^0. Any suggestion how to do? Is the Ct value higher than 35 reliable? If it's not reliable, how to deal with my unknown samples with higher Ct Value(more than 35)?
I need help very much!Look forward to your answer.
Ideally the Ct should be lower...
how long is your amplicon? Were the primers developed for QPCR?
Did you optimise your annealing temperature, etc?
Generally, there are a series of optimisations to do before the actual experiement, like determining the best primer pair, the best anneal temp, and so on.
I am not sure what you mean by 5.9*10^0. I believe this equals 5.9. Your standard curve should have a range of copy numbers from 1 up to 100000 or so, or thereabouts. I can't tell how you are running your standard curve.
Thanks for Patty's reply.
My amplicon is 154bp. I use plasemid inserted the target amplicon as standard. The original concontration is 100ng/ul, I calculated the my standard as 5.9*10^11 copies/ul. Then I did serial 10 fold dilution and use the range of 5.9*10^5 copies/ul to 5.9*10^0 copies/ul to make the standard curve. I add 7.5 ul per 20 reaction volume.
The primers(Tm 60 degree) and probe(Tm 66 degree) were disighed by company for qPCR and synthesized by Operon company.
I optimized the ratio of primer(0.1uM-0.5uM) and probe(0.05uM-0.5uM) and chose the best one. According the annealing temperature, there is no obvious difference between 58-62 degree.
If I still can't get signal from 5.9*10^0 copies/ul, can I use the standard curve to calculate the copy number of unknown samples which Ct value was higher than that of 5.9*10^0 copies/ul ? Is that permitted?
Must I make the standard curve in every experiment? If I fix all the reagents and all the PCR conditions, can I use the same standard curve in different run?
Don't laugh at my stupid question, Pls.
I need suggestion very much.
I seldom believe qPCR data with CT value over 35 unless I nwo the amplification efficiency is low, The CT value of reaction withr target DNAs/cDNAs near to 10 copies usually showing big variation.
I suggest you to increase template amount in qRT-PCR for your tested samples...
Dear Rve,
Sure I tried to increase template amount of my unknown samples up to 200~500ng/per reaction, but it can't help too much. Now I use about 100ng/reaction, even I increase the amount 10 times (1ug) , the Ct value can increase about 3 cycles, but for the reaction itself, the PCR will be inhibited very much!
I think in my case, most of my samples contain only several target copies per ng, for so low target copies, if I still want to increase the Ct value, How to do.....
So now I have two conflict probelem: one is I can't get the highest Ct point in my standard curve(5.9*10^0 copies/ul), another one is I can't decrease the Ct value of my unknown samples(more than 35 cycles)......
Urgent help!