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-   -   Doubt with melting curve qPCR (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/26332-doubt-melting-curve-qpcr.html)

mjhr 10-30-2008 02:36 PM

Doubt with melting curve qPCR
 
1 Attachment(s)
I have a doubt with my results in the qPCR using SYBR Green, and I do not find the reason of it. Maybe do you know:
1) Why in the melting curve for a same gene are there two different peaks? They are rather joined, but there are two.
2) Can she consider that her results are ok with this difference in the peaks? There are 2 peaks for the house keeping gene, but also for the gen of interest.

Please find attached the graph:

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Thank you in advance,

Maria

Aga 11-13-2008 04:30 PM

Re: Doubt with melting curve qPCR
 
One of the reasons of the slightly different peaks could be the fact, that SYBR Green is non-specific and intercalates into dsDNA not every single base but in some intervals which we can't actually control. The intervals may be a little different and that causes little differences between samples, thus this may affect the peaks.
The resolution of SYBR Green, in my opinion, could be also the cause. The peaks having Tm differing from each other in less than 1-2 degrees Centigrade may be indistinguishable.

I would interpret the above peaks as the presence of the same gene.

mjhr 11-21-2008 04:35 PM

Re: Doubt with melting curve qPCR
 
Thank you very much for your opinion and explanation. I run the PCR product in gel and they are at the same distance.

Aga 11-21-2008 07:06 PM

Re: Doubt with melting curve qPCR
 
This may be true, because gels have even lower resolution than SYBR Green in a melting curve.
I had one situation that after I amplified one DNA fragment I had two samples between which Tm of a product in a melting curve differed in 3 degrees, which was highly distingiushable in a melting curve, obviously.
But I ran those real-time PCR products in a gel and there was no difference between bands sizes. It seems that with increased accuracy of a method you start to get more questions...

rohanpatil 10-03-2009 05:45 PM

Re: Doubt with melting curve qPCR
 
2 Attachment(s)
I have a doubt regarding melting curves. I have attached 2 images.

1) For Gene 1 the 2 peaks for control are slightly lower as compared
to the 2 peaks of sample. (The temperature of peak is same for both
controls and samples)
2) For gene 2 the 2 peaks for control and 2 peaks of sample are almost
the same. (again the temperature of peak is same for both controls and
samples)

Question: I know for one gene the temperature of peak should be same, however is it necessary that the peaks should overlap in terms of height?
The other way of asking it is what does the height of melting curve peak indicate?
I am using SYBR green and MX3000P for analysis.

Thanks

Aga 10-04-2009 02:10 PM

Re: Doubt with melting curve qPCR
 
The photos you sent are very low quality and not much is seen.
Lower peak height indicates that less product is amplified, i.e. the higher the peak the higher the amount of amplicon. I'm noit sure whether this relation is strictly linear.

Higher peak can mean that there was higher concentration of target DNA at the beginning or there was higher PCR efficiency (if you finish the reaction before it reaches plateau phase). Off course lower peak is the result of the opposite.

With SYBR Green I without reference dye and internal amplification standard the height of the peak doesn't give you any information you can actually use for quantitation.

hp10aaa 06-25-2010 10:01 AM

Re: Doubt with melting curve qPCR
 
Can I know what should be the mininum height of Tm in RT-pcr. am getting around 2000 is it fine or do i need to normalise the Tm till I get the maximum Tm value?

televisiononcomputer 06-25-2010 01:18 PM

Re: Doubt with melting curve qPCR
 
I'm not sure either .. somebody please clarify

Aga 06-26-2010 07:56 AM

Re: Doubt with melting curve qPCR
 
You do not normally normalize Tm peak height in SYBR Green I real-time PCR. This is because you don't, nowadays, rely on peak height for quantification of your DNA in a sample - this is done by using probe-based assay - it is far more accurate.

If initial target DNA concentration in your samples is identical and, using the same reaction conditions, you have different Tm peaks height for these samples it may mean PCR efficiency differ between the samples. However, this is best seen on amplification fluorescence curve. Ideally, no matter what is the initial target DNA concentration in your samples, the fluorescence curves should be parallel during the exponential phase. If not you may have some PCR inhibitors and/or not enough purified DNA. To compare two samples it is important that PCR efficiency is the same.

hp10aaa 06-27-2010 09:00 PM

Re: Doubt with melting curve qPCR
 
Thank you !!!
1.May I know how to normalise the house keeping gene for a single cell type...

2. what are the different sets of normalising required befor going to actual comparison of epression depending on treatment of cell systems....


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