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Erratic Cq values from Plasmids

plasmids qPCR Cq stability

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#1 Montys

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Posted 21 April 2021 - 06:44 AM

Hi there,

 

I do qPCR analysis and we designed some positive controls (PC) using fragments of pathogens in plamids. These plasmids are purified out of bacterial cultures.

 

 

If we start with our qPCR we get a certain Cq for a specific concentration of the PC. After some time the Cq drops more than 3-9Cqs for the same concentration.

 

Surprisingly after almost half a year the Cq regains the value of the original Cq value from a freshly made PC with the same plasmid concentration.

 

eg.:

 

Day 0     25 Cq

Day 40   29 Cq

Day 90   29 Cq

Day 180 25 Cq

 

 

Does anybody have an idea how this can happen. We aliquoted the freshly made PC and directly without any further dilution used it for PCR reactions. That would be much appreciated.

 

Best regards

Andreas



#2 bob1

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Posted 21 April 2021 - 02:29 PM

Sounds like a degradation problem - your aliquot is undergoing degradation as you use it. This could be from a number of things, but the most likely are lack of buffering in the plasmid prep leading to acid hydrolysis of the DNA or freeze/thaw cycles causing physical damage to the DNA.



#3 Montys

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Posted 21 April 2021 - 10:05 PM

Sounds like a degradation problem - your aliquot is undergoing degradation as you use it. This could be from a number of things, but the most likely are lack of buffering in the plasmid prep leading to acid hydrolysis of the DNA or freeze/thaw cycles causing physical damage to the DNA.

 

Oh sorry I think you misunderstood me.

 

We use each aliquot only once. All aliquots were frozen at the same time under equal conditions. So if there is degradation, only some aliquots would have it and others not. This is very unlikely, because all aliquots have the same amount of DNA in the same buffer. 

 

The concentrated PC was made at day 0 from same plasmid extraction with same buffer:

The PC was then diluted up to the desired concentration. Afterwards aliquots with same volume were prepared into 4 vials which are my used Aliquots (AL). 

 

On each measurement day one Aliquot was thawed and PC was measured in triplicates:

Al 1: Day 0     25 Cq

Al 2: Day 40   29 Cq

Al 3: Day 90   29 Cq

Al 4: Day 180 25 Cq

 

 

Hope this clarifies everything.

 

Best Regards



#4 Gonzalez

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Posted 22 April 2021 - 06:43 AM

Have you checked your plasmid fragment for secondary structure (like hairpins and their melt temperatures)? It could potentially be due to that. A more likely explanation to me is that it's your other reagents. Your Cq (Ct) value will go up as the qPCR becomes less efficient due to old reagents, then goes back down when you open a new vial of polymerase.



#5 Montys

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Posted 23 April 2021 - 06:34 AM

Have you checked your plasmid fragment for secondary structure (like hairpins and their melt temperatures)? It could potentially be due to that. A more likely explanation to me is that it's your other reagents. Your Cq (Ct) value will go up as the qPCR becomes less efficient due to old reagents, then goes back down when you open a new vial of polymerase.

Hi Gonzales,

 

Sadly the other reagents seem to work perfectly. I used a WHO Standard and patient material as well. Over the whole time the SD of Cq values for the standard as well as the patient material was only around 0.16. 

 

Here is one example of 4 measurements of a WHO Standard dilution:

First day was only a duplicate measurement all other had triplicates.

 

26.01 25.96   25.68 25.71 25.72 25.79 25.61 25.80 25.79 25.61 25.80

 

So maybe it has something to do with the plasmid structure yes, but I still wonder why it is sometimes an issue and sometimes not? 

 

Andreas



#6 Montys

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Posted 28 April 2021 - 01:09 PM

Hi,

 

sorry one could not edit a post.

 

I wanted to add that we always use one time aliquots for all substances including the enzyme. So definetly it is something with the plasmid. Maybe sometimes we didn't waited as long as other times before we pipetted everything together.

BEst,

Andreas

 

 

 

Have you checked your plasmid fragment for secondary structure (like hairpins and their melt temperatures)? It could potentially be due to that. A more likely explanation to me is that it's your other reagents. Your Cq (Ct) value will go up as the qPCR becomes less efficient due to old reagents, then goes back down when you open a new vial of polymerase.

Hi Gonzales,

 

Sadly the other reagents seem to work perfectly. I used a WHO Standard and patient material as well. Over the whole time the SD of Cq values for the standard as well as the patient material was only around 0.16. 

 

Here is one example of 4 measurements of a WHO Standard dilution:

First day was only a duplicate measurement all other had triplicates.

 

26.01 25.96   25.68 25.71 25.72 25.79 25.61 25.80 25.79 25.61 25.80

 

So maybe it has something to do with the plasmid structure yes, but I still wonder why it is sometimes an issue and sometimes not? 

 

Andreas

 


Edited by Montys, 28 April 2021 - 10:23 PM.






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