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I need an urgent help with q-pcr amplification plots


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11 replies to this topic

#1 genom38

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Posted 28 October 2010 - 11:25 AM

Hello everyone,

I have done q-PCR for a while and I have problems with the amplification curves. I have got weird amplification plots with zigzag curves and I cannot find the reason. I use Roche SYBR green master mix and specific primers. When I use same SYBR green master mix to amplify another product I get normal amplification curves, therefore I do not think that the problem is master mix (Differences are cDNA and primers only, even the conditions are same). Here is an attached sample of amplification curve. Have you ever seen an amplification curve like this? What can be the reason?

Attached Files



#2 gangut

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Posted 01 November 2010 - 04:04 AM

Hi,

isn't the gene you're amplyfying a very low-copy one? Perhaps you should try taking more cDNA. Is there any amplification signal in water?

#3 genom38

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Posted 04 November 2010 - 10:53 AM

Hi,

isn't the gene you're amplyfying a very low-copy one? Perhaps you should try taking more cDNA. Is there any amplification signal in water?


Sometimes lower concentration of cDNA or diluted cDNA works better than higher amounts of cDNA, I think it's because of some interference coming from RT reaction. I do not think that this situation is related with cDNA amount but cDNA or primer quality. However, I think primer quality is ok because melt curve is good enough. There is not any amplification signal in no template control (in rxn mix-water), the grey straight line at the bottom belongs to no template control.
I could find interpretations about hook-shaped amplification curves, but I could not find any information about zigzag-shaped amplification plot in the literature. For this reason, I need your ideas...

#4 nightingale

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Posted 04 November 2010 - 03:00 PM

Hello There,

have a look at this guide Attached File  QPCR_&_QRT-PCR_Troubleshooting_Guide.pdf   76.17KB   415 downloads
esp. the last two raws ...

hope beneficial in a way or another..

Best Wishes,
nightingale

" The more you learn, the more you realize how little you know ... "

#5 genom38

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Posted 08 November 2010 - 07:38 AM

Hello There,

have a look at this guide Attached File  QPCR_&_QRT-PCR_Troubleshooting_Guide.pdf   76.17KB   415 downloads
esp. the last two raws ...

hope beneficial in a way or another..

Best Wishes,
nightingale



Hello Nightingale,

Thank you for the link. The last problem is related to my problem, however I could not understand how to solve. Do you know what "the moving average data correction algorith" is? How can I find this algorithm, is it inside the software?

#6 UBClabbie

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Posted 08 November 2010 - 01:47 PM

I think its likely that your cDNA sample is of poor quality. Try cleaning up your samples.
Another thought is that your mastermix is no good anymore.
Try a fresh batch? Freeze thaw can really affect the mastermix's stability and cause problems in your amp plots

#7 nightingale

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Posted 08 November 2010 - 03:55 PM


Hello There,

have a look at this guide Attached File  QPCR_&_QRT-PCR_Troubleshooting_Guide.pdf   76.17KB   415 downloads
esp. the last two raws ...

hope beneficial in a way or another..

Best Wishes,
nightingale



Hello Nightingale,

Thank you for the link. The last problem is related to my problem, however I could not understand how to solve. Do you know what "the moving average data correction algorith" is? How can I find this algorithm, is it inside the software?


you are most welcome Genom38,
actually am not in the lab, to check the software ...
i think it will be there ...
read the catalog, or ask the vendor about it,
any how i have found you this :

Prikl Biokhim Mikrobiol. 2006 Sep-Oct;42(5):520-8.

[Real-time PCR: approaches to data analysis (a review)]

[Article in Russian]

Rebrikov DV, Trofimov DIu.

Abstract

The registration of the accumulation of polymerase chain reaction (PCR) products in the course of amplification (real-time PCR) requires specific equipment, i.e., detecting amplifiers capable of recording the level of fluorescence in the reaction tube during amplicon formation. By the time the reaction is completed, researchers obtain DNA accumulation graphs. The review discusses the most promising algorithms of analysis of real-time PCR curves and possible errors, whether caused by the software used or the operators' mistakes. The data included will assist researchers in understanding the features of the method to obtain more reliable results.

PMID: 17066950 [PubMed - indexed for MEDLINE]

seems promising, BUT be aware of the written language ...
All The Best ...
keep us on track ...

" The more you learn, the more you realize how little you know ... "

#8 genom38

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Posted 09 November 2010 - 11:54 AM

I think its likely that your cDNA sample is of poor quality. Try cleaning up your samples.
Another thought is that your mastermix is no good anymore.
Try a fresh batch? Freeze thaw can really affect the mastermix's stability and cause problems in your amp plots


Hi Biotechgirl,

I perform another PCR for another gene at the same time using same mastermix and its amplification plot is ok. I thought the same things with you and I made new cDNA with renewed RT, buffer, water... I tried the same q-pcr with new cDNA-old SYBR green (which I did not use before), new cDNA-new SYBR green (which I used for this gene of interest), old cDNA (problematic)-old SYBR green in the same reaction. The amplification plots were again jagged! And also, all the time, before q-PCR I try to optimize conditions with normal RT-PCR. There was no problem with the products of this gene, and also melt curves of q-pcr are again good; hence I think that there is no problem about my primers.
In summary, it seems that there is no problem about
* primers
* cDNA reaction (reverse transcription)
* SYBR green

I am again complicated! Where is the problem?! :(

#9 genom38

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Posted 09 November 2010 - 11:58 AM



Hello There,

have a look at this guide Attached File  QPCR_&_QRT-PCR_Troubleshooting_Guide.pdf   76.17KB   415 downloads
esp. the last two raws ...

hope beneficial in a way or another..

Best Wishes,
nightingale



Hello Nightingale,

Thank you for the link. The last problem is related to my problem, however I could not understand how to solve. Do you know what "the moving average data correction algorith" is? How can I find this algorithm, is it inside the software?


you are most welcome Genom38,
actually am not in the lab, to check the software ...
i think it will be there ...
read the catalog, or ask the vendor about it,
any how i have found you this :

Prikl Biokhim Mikrobiol. 2006 Sep-Oct;42(5):520-8.

[Real-time PCR: approaches to data analysis (a review)]

[Article in Russian]

Rebrikov DV, Trofimov DIu.

Abstract

The registration of the accumulation of polymerase chain reaction (PCR) products in the course of amplification (real-time PCR) requires specific equipment, i.e., detecting amplifiers capable of recording the level of fluorescence in the reaction tube during amplicon formation. By the time the reaction is completed, researchers obtain DNA accumulation graphs. The review discusses the most promising algorithms of analysis of real-time PCR curves and possible errors, whether caused by the software used or the operators' mistakes. The data included will assist researchers in understanding the features of the method to obtain more reliable results.

PMID: 17066950 [PubMed - indexed for MEDLINE]

seems promising, BUT be aware of the written language ...
All The Best ...
keep us on track ...



Hi again Nightingale,

I have tried to communicate with vendor, I hope they will answer.
Thank you again for the review, but I couldn't reach the fulltext unfortunately .

#10 nightingale

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Posted 10 November 2010 - 11:05 AM

Hi again Nightingale,

I have tried to communicate with vendor, I hope they will answer.
Thank you again for the review, but I couldn't reach the fulltext unfortunately .


hello Genom38,

hope you have heard s.th from the vendor ...
i have found this : technical note
read PCR reaction optimization # 5.

Best Wishes.
" The more you learn, the more you realize how little you know ... "

#11 genom38

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Posted 14 November 2010 - 01:27 PM

Hi all again,

I think I found the reason of the weird amplification plot :D I considered the technical note #5 in the Bio-rad's HRM-tech note paper which is suggested by Nigntingale. There was an efficiency problem in my experiment because, the fluorescence remained at low levels and efficiency calculated from standard curve was low also. In the tech-note #5 it is proposed that jagged-plots could be the result of a poor amplification. Therefore, I decided to decrease the annealing temperature. First, I decreased temperature from 62 to 60 and got smoother plots. Then I decreased 2 degrees more and set the annealing temperature to 58 and I got smoother and smoother plots. Also fluorescence increased up to 100 from 20s and efficiency increased to 100%. I understood that the problem was annealing temperature. I will continue with this annealing temperature, I hope there will not be a problem in the melt curve for the oher samples those were not included during optimization.

I want to thank Nightingale very much for the effective references and the others for all the contributions. I hope this discussion helps all of you.

#12 nightingale

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Posted 15 November 2010 - 11:06 AM

Hi all again,

I think I found the reason of the weird amplification plot :D I considered the technical note #5 in the Bio-rad's HRM-tech note paper which is suggested by Nigntingale. There was an efficiency problem in my experiment because, the fluorescence remained at low levels and efficiency calculated from standard curve was low also. In the tech-note #5 it is proposed that jagged-plots could be the result of a poor amplification. Therefore, I decided to decrease the annealing temperature. First, I decreased temperature from 62 to 60 and got smoother plots. Then I decreased 2 degrees more and set the annealing temperature to 58 and I got smoother and smoother plots. Also fluorescence increased up to 100 from 20s and efficiency increased to 100%. I understood that the problem was annealing temperature. I will continue with this annealing temperature, I hope there will not be a problem in the melt curve for the oher samples those were not included during optimization.

I want to thank Nightingale very much for the effective references and the others for all the contributions. I hope this discussion helps all of you.


first : i would like to thank you for your nice words ...
am overwhelmed by your nice message :)
thank you ...

second to that : am really glad that by referring to my suggested, your matter has seen light out there :D

Wishing You All The Best

" The more you learn, the more you realize how little you know ... "




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