I get no amplification but I see a very high fluorescence (normal ROX profile) already before the first PCR cycle (raw data: 1x10^6 fluo units). Such a high fluorescence declines rapidly during the dissociation step after the PCR (melting stage), therefore I think it could be contaminant DNA. However, I don't know how it could be, as my RNAs undergo extensive digestion in the presence of DNase I.
Does anyone know what kind of problem I am facing?
Please, note that just some samples present this problem, whereas many others do not. Moreover, I always set up master mixes, thus I know my problems do not arise during RT-PCR steps, suggesting a possible random contamination before RNA extraction (contaminant DNA too high for DNase treatment?) or after the incubation with DNase.
Thanks a lot in advance.
Stefano Falone
Did you measure 260/280 & 260/230 for your RNA samples? It could be possible that you have phenol contamination that is being carried forward and giving you high background. Other possibility is primers that you are using, do you have same problem when you trying to amplify house keeping gene during real time PCR?
High fluorescence that disappears with the first melt step does rather sound like DNA contamination.
Two things you could check if you think it's contaminant genomic DNA: run your samples on an agarose gel (look for high mol wt bands that barely enter the gel), or just perform a melt curve on the samples (i.e. add your sybr green mix, but don't run any amplification, go straight to a melt curve). Genomic DNA should give a relatively broad, fairly high-temp melt peak.
Also, if you have any primers that work with genomic sequences (genotyping primers, for instance) and that bind to intronic sequence, you could try using those on your samples.
Of more concern is "I get no amplification": what does this mean? As in, you get no product from these reactions at all?
One picture is better that 1000 words :)
As You can seen, my fluorescence plot shows very high fluo background (normal ROX), as if contaminant DNA were present. At the beginning of the melting curve (dissociation stage), fluorescence drops. Please, take a look at the file attached.
Thanks a lot in advance.
Stefano
I'm having trouble understanding the X-axis: it says 'cycles', but that would imply that your fluorescence doesn't drop till cycle 42 or so, and (more worryingly) that you're running 133 cycles.
If for "cycles" I should be reading "degrees C of a melt curve", then it looks like you have an enormous amount of something that melts very easily, and a small amount of stuff that melts relatively late, but frankly the fact that the curve literally drops sharply (rather than sloping gently) at the 43 point tends to suggest it's not dsDNA strand separation.
I'm wondering if it's some sort of fluorophore carryover from your oocyte preps. If you take these samples and run melt curves with no sybr green present (i.e. just cDNA and water, no mastermix) do you no longer see this effect?
My aspecific fluorescence starts dropping after the last PCR cycle (#40), right at the beginning of the dissociation stage, when T starts to rise. No fluorophore is added to the oocytes, neither does the oocytes contain any fluorophore per se.
I will try to run samples w/o SYBR green.
Thx a lot
Stefano
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