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Low efficiency - standard curve

Low efficiency - standard curve - Real-Time PCR and Quantitative PCR Forum

Low efficiency - standard curve - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 05-29-2009, 01:12 AM
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Default Low efficiency - standard curve



Hi, Iím having troubles with my standard curve. I'm using DDCt method for relative quantification. Iíve already performed qPCR with other genes and had an excellent efficiency. But now Iím working with weakly expressed genes. Iíve already used both cDNA and purified PCR products, in 10x and 5x dilutions, the amplification plots seem to be perfect, regular distances between the dilution points, good correlation, but when I analyze the standard curve slope is between -4.20 and -4.08, and of course, low efficiency. There are no primers dimmers, no contamination cause NTC is OK. I donít know what is happening. Could somebody give me a suggestion? Thank you.
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Old 03-15-2010, 10:15 AM
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Default Re: Low efficiency - standard curve

PCR inhibitors? different source of RNA or isolation method?
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Old 04-09-2010, 09:29 PM
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Default Re: Low efficiency - standard curve

Have you tried reducing the annealing temperature - say 2oC?
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Old 04-12-2010, 04:26 PM
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Default Re: Low efficiency - standard curve

Have you tried redesigning primers? Maybe there's something wrong with your primer set that is non-obvious. In my experience, occasionally a primer set will just not work very well for no apparent reason.
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Old 10-14-2010, 06:54 PM
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Default Re: Low efficiency - standard curve

I got a similar situation for my 5x dilution series: distances between the dilution points is the same (1,60 delta CT) for the first four dilutions but still get a slope of -4,837 and an amplification of 61% (calculated with the E=[10^(-1/slope)]-1 - formula).
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Old 10-07-2011, 05:30 AM
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Default Re: Low efficiency - standard curve

would it be possible if you show us the amplification plot? and the standard curve as well thanks!
Can you supply with information below;
1) Are you using commercial Real-Time PCR kit or home made?
2) GC content and size of your amplicon.
3) Concentration of your template DNA.
4) Melt curve of that experiment.
BTW, what Real-Time machine are you using?
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