Hello there, I am currently working in an absolute quantification with RT-qPCR. I have a synthetic standard of 150 pb approximately. I have made 1:10 dilutions with the same standard but when I see the results displayed on the equipment, the Ct are similar in all my dilutions, they don’t achieve 3.3 cycles of difference between one dilution and other. Furthermore, I have tried with a positive sample, with this one, the results are good but not with the standard.
For example:
Dilution_2e7 copies/ul the Ct value is 20.43
Dilution_2e6 copies/ul the ct value is 21.59
Dilution_2e5 copies/ul the ct value is 21.61
Dilution_2e4 copies/ul the ct value is 21.38
Can you please help me to understand what can be happening?
1) It could happen that contamination or impurity has been introduced in your (standard) reagents.
If this contamination has inhibiting effects on enzymes, such effects are mitigated by dilutions, so that you could even obtain earlier Ct values than in the less diluted samples.
Broadly, try to think about possible limiting factors effective at higher concentrations (and less effective in diluted conditions).
2) Anyway, a distance of 3.3 cycles between dilutions could be not to be rejected...
...and 3) don't forget to take a look to melting curves... usually they reveal a lot about such troubles...
I think 3.25 difference is what you need for 1:10 dilutions but yours are way off.
Test your primers on a different sample first to see if the primers are working well.
I agree with both. If your synthetic standard is not contaminated than probably your primer has low efficiency and should be redesigned. Run a standard curve with a cell extract to calculate primer efficiency and also run some in silico analysis on your primer sequences to check possible hairpins and dimers. As Amilcare mentioned, if you are using SYBR check your melt curve, it can help you confirm primer dimers.
Dear Luz Maria Sanchez !
Just wondering, if you solved your problem?
I'm experiencing a similar problem right now. The only difference is that it actually worked before already! I had really nice curves for my standard dilutions with about 3.3 cycles difference as mentioned above There was no signal in the -ve controls and the melt curves showed that there was only the DNA I was looking for. Then I used new primers from another company and it did not work anymore. The contribution of the Ct values is like you described yours.
I now ordered new primers (from the old and the new company), I use the same concentration, the same conditions (already varied the annealing temperature again), freshly prepared standards and nothing works anymore!! And yes, I already tried new DNA as well.
Did you find a solution which could actually help me as well?
Nadine Barna Have you found any solution to this problem? It's also happening to me. I use positive control in my run, and the CTs are the same for different dilutions.
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