4 replies to this topic

[skolyar]

Hi all,

Possibly someone can explain this to me...

I need to prepare some serial dilutions of standards for generation of standard curves, which will be later used to estimate abundace of 4 genes in complex environmental samples using qPCR. I have cloned these 4 genes into plasmids, purified and estimated concentration of the isolated plasmid DNA using a Nanodrop (spectroscopy) and Qubit (fluorometry). I normalised the concentration for each plasmid,prepared 10 log dilutions and ran qPCR with them. I have observed, however, that Ct values for standards containing the same estimated concentration of the template(e.g. 10E8 copies/ul) for each gene were significantly different. The difference was up to 6 cycles. The amplification efficiency was similar (1.90-1.94). Gel analysis of the plasmid DNA showed a single band (no signs of RNA or bacterial genomic DNA contamination). The same machine and PCR mix was used in all reactions.

Can anyone explain such great discrepancy?

Thanks

Edited by skolyar, 21 November 2010 - 06:03 PM.

[ElHo]

Could be due to the different lenghts of your different amplicons. The longer an amplicon, the more SYBR will intercalate, the brighter the fluorescence will be. So it´s not as simple as identical copy numbers resulting in identical CT values.
Concerning normalization, are you sure you normalized your different plasmids to equal molarity and not equal mass?

[skolyar]

ElHo, on 23 November 2010 - 04:10 AM, said:

Could be due to the different lenghts of your different amplicons. The longer an amplicon, the more SYBR will intercalate, the brighter the fluorescence will be. So it´s not as simple as identical copy numbers resulting in identical CT values.
Concerning normalization, are you sure you normalized your different plasmids to equal molarity and not equal mass?

Thanks ElHO!
I thought it might be the case. Would you expect the difference in the fluorescence (and Ct value)to be proportional to the amplicon size? For instance, would a 2-fold difference in the the amplicon size result in the ~2-fold difference in the fluorescence and ~1 Ct value shift, for the same concentration of the template at the same conditions (assuming the optimum amplification efficiency)?
Appreciate your help.

Edited by skolyar, 23 November 2010 - 12:19 PM.

[skolyar]

ElHo, on 23 November 2010 - 04:10 AM, said:

Concerning normalization, are you sure you normalized your different plasmids to equal molarity and not equal mass?

Yes, I did convert all the concentrations into copies/ul.

Thanks

[ElHo]

skolyar, on 23 November 2010 - 12:18 PM, said:

ElHo, on 23 November 2010 - 04:10 AM, said:

Could be due to the different lenghts of your different amplicons. The longer an amplicon, the more SYBR will intercalate, the brighter the fluorescence will be. So it´s not as simple as identical copy numbers resulting in identical CT values.
Concerning normalization, are you sure you normalized your different plasmids to equal molarity and not equal mass?

Thanks ElHO!
I thought it might be the case. Would you expect the difference in the fluorescence (and Ct value)to be proportional to the amplicon size? For instance, would a 2-fold difference in the the amplicon size result in the ~2-fold difference in the fluorescence and ~1 Ct value shift, for the same concentration of the template at the same conditions (assuming the optimum amplification efficiency)?
Appreciate your help.

Good question!  Maybe sequence context also plays a role in how much SYBR can intercalate and how intense the fluorescence will be. But I´m not sure about this.