It is a real-time quantification of RNA targets with QuantiFast Probe RT-PCR kit (Qiagen) using TaqMan probe. I would like to know what this "negative curve" means?
Hello Leandro, it is not really a negative curve, the software of your RT PCR platform will always take the starting signal as a base and the increase as a "real" amplification.
If you have to much DNA contamination the software will detect all of these double strands fluoresence signals as background, and after a few cycles of increase you will have a decrease because the sybr green will rather go to the short amplicons than to the total genomic DNA contamination, having "less" signal than the beggining.
you should run a electrophoresis of your starting material, if its any band or smearing above the RNA bands would mean you have too much DNA and you need more DNAse steps or less biomass for your extractions!
I know this cuz I suffer from this too.
best of luck!
Hi Leandro
I have seen curves like yours when I have analysed samples with too much template in. The software in most PCR machines analyses a set of cycles early in the reaction to set the background (ie this is how much background fluorescence we see when no significant fluorescence is being generated) and then subtracts that from anything generated subsequently in the reaction. The default for most machines is something like cycles 3-15 as typically fluorescence isn't generated here unless far too much template is used....however, it looks like you have very positive samples with amplification in this window which then distorts the curves.
You can manually alter the background parameters in most software - if you alter this your curves will magically turn into traditional sigmoidal curves when reanalysed (I imagine you'll need to change it to cycles 3-5 as you have so much template).
If you can't alter the parameters, repeat the PCR but dilute your samples 1:1000 or possible more...that will have the same effect. A 1:10 dilution results in a shift of 3.32 cycles, so 1:1000 will shift the curves 10 cycles - your samples have a Ct of about 10 so a 1:1000 dilution will shift it to ~cycle 20
All the best
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