Hi all,
Do anyone know what does the Peak height means in Melt curve ( Y axis derivative reporter) ?Initially, I thought the peak height corresponds to the Sybr green fluorescence intensity but I am not sure if that is the case.
Note: The samples are the same ( Same RNA= similar melting temp( Tm)) but just different peak height.
Dear Yap yi jun
After the amplification, SybrGreen is in between the two strands of the double helix. Raising the temperature, you will get a point where 50% of the amplicons will melt and the SybrGreen will be release into the medium. SybrGreen emits fluorescence when inside double stranded DNA. Therefore, when it is released the fluorescence level strongly decreases.
The derivative of the function “fluorescence vs. Tmelting” (dF/dT) represents the rate of the fluorescence variation in the reaction. The graph you see is” -dF/dT vs. Tmeting” and it represents the fluorescence variation as a pick.
Regards
Eugénia
Hi Yap yi jun
I believe the height of a melting curve peak is partly related to the amount of product.and also to late amplifications. DIference in height may also be associated to polymorphisms.
Hi Yap yi jun
Peak height in melt curve refers to the amount of fluorescence signal, which means a higher peak indicate more products in polymerase reaction than a lower peak. It's just like a normal PCR which some bands may be fainter than the other.
Dear Yap yi jun
After the amplification, SybrGreen is in between the two strands of the double helix. Raising the temperature, you will get a point where 50% of the amplicons will melt and the SybrGreen will be release into the medium. SybrGreen emits fluorescence when inside double stranded DNA. Therefore, when it is released the fluorescence level strongly decreases.
The derivative of the function “fluorescence vs. Tmelting” (dF/dT) represents the rate of the fluorescence variation in the reaction. The graph you see is” -dF/dT vs. Tmeting” and it represents the fluorescence variation as a pick.
Regards
Eugénia
Hi, in your case both the peaks are lying in almost same temperature range while only differ in their heights. One possible explanation of your image could be that the sample amount differs in your duplicates and this is very likely due to pipetting error, or some evaporation before the qPCR run from your plate has happened due to ambient temperature you are working or may be some delay in sealing your plate finally. It can also happen, when sealing of the plate is not proper and we get some evaporation loss during the run inside the thermal block. Before pipetting your samples into the plate try to do a brief spin down of your samples always so to get a uniformity in your cDNA sample.
- Badges
- Science topic