Strange problems with B-actin amplification - (Oct/20/2006 )
We use to perform a b-actin PCR with "LightCycler- FastStart DNA Master SybrGreen I" and we get a CP around 13-15. We made RT reaction with 1ugr RNA (final volume 20ul) and we use 2ul for capillaries in PCR. We extract RNA with trizol method or with Nucleospin RNAII "MN".Usually, we don't have problems with this setup.
In the last two weeks, the amplification curves from new samples have a lower slope and a CP near 26.
We perform some experimetns to explain this:
- The standards works well.
- We perform the retrotranscription with another enzime and works bad (CP=26).
- we diluted one of the samples that works bad, and the result was: pure cDNA CP=26; 1:10 cDNA CP=18; 1:100 cDNA CP=21.5.
Because of these results, we suspect that there is some inhibition in the samples (that appears now!!!).
Attached you can see amplification curves of samples and standards. Also you can see my standard curves.
What is going on? Any idea???
Thanks a lot!!!
Try diluting your cDNA several (4-5) log folds. It seems that you Ct value starting around 10 which is suggestive of two things: (1) your probe is picking up other than your gene (i.e. b-actin) (2) check you threshold baseline. Normally you would have a 2^10 fold difference between priming and signal. If need, adjust you threshold baseline to 2 to 7. But, then it is to small to set a correct background signal.
Be aware of pitfall of comparing b-actin at one or two dilutions. Ideally, one would make a plot of b-actin dilution before comparing ddCt to other gene.