Do I understand right: when running qPCR with SYBR green detection, it is important that two amplicons (GOI and reference gene) should be of the same lenght ?
Once It is supposed to be relative copy number assessment, using delta-deltaCt formula by Livak et al. - the fluorescent signal will be detected at the certain Ct, so if two amplicons are different in length, (e.g. the same quantity of amplicons will give different signal intensity) we cannot use them for relative quantification. - is that right?
Thanks for the comments!
Test your primers for their efficiency before running the experiment. That is, set up a calibration curve of serially 10x diluted standard DNA and plot the resulting Ct (y-axis) vs. Log(10) of target copy number (x-axis). The slope should be as close to -3.32 as possible (ie. 3.32 doublings make a ten-fold amplification). If both primer sets are highly efficient, don't worry about amplicon size.
No, you should be looking at rising points (Cts) not the RFU values.
The rising point (Ct) is mainly determined by template initial abundance, hence more abundant templates will give earlier Cts (not because they are longer templates leading to brighter signal).
---- When running qPCR with SYBR green detection, it is important that two amplicons (GOI and reference gene) should be of the same length?
No, that is not necessary. However, to get best results you should design your primers to amplify products optimally within 100-150 bp in length and should not exceed 500 bp. For example, you can make amplicon size of 100 bp for GOI and 150 for reference gene.
---- So if two amplicons are different in length, (e.g. the same quantity of amplicons will give different signal intensity) we cannot use them for relative quantification?
If I understand your question in the correct way, this statement is correct for the first part (if two amplicons are different in length, the same quantity of amplicons will give different signal intensity). However not true for the second part (we cannot use them for relative quantification).
Basically, you are not comparing signal of GOI with signal of reference gene, rather you compare signal of GOI across several samples, and then compare signal of reference gene across same samples. The purpose of measuring reference gene is to normalize the quantity of your template across samples, and has nothing to do to compare with GOI.
For that reason you should not worry if amplicon size is different between reference gene and GOI.
Also, I would like to note: even if the amplicon product size is same (for two different templates), that doesn’t mean necessarily they produce same signal intensity. Mainly for two reasons:
1) GC content may be different
2) Amplification efficiency for each of them may be different.
Thank you all for your answers!
Totally agree with Can and Mohamed, that basically we compare Ct's but not the signals strength. So all three methods of relative quantification (Livak, Pfaffl, and deltaCt) are based on slightly different equations with Ct. But, at the same time, Ct depends on the signal strengh, doesn't it ? - The stronger the signal the smaller the Ct value.
Thus, considering that the amplicon's lengh is proportional to the amount of SYBR green trapped (e.g. signal strengh) - it might be assumed that after all, when calculating E^(Ct_ref. gene - Ct_target gene) we can get the different values for two different amplicons (of different length) of the Ref. gene, because the Ct is different.
Sorry, if this issue is confusing, and I ran qPCR all the times with a bit different amplicons length, which didn't likely affect the results. But it occured to me that it might bias the results. And before I try to do with two different amplicons of ref.gene, I just wanted to ask your opinion.
Thank you for your comments!
Amplicon size isn't that important, remember you are trying to measure Cts not RFUs. And Cts are primarily determined by template abundance. Typical guidelines for primer design use 75-150 bps for SYBR green chemistry so if you stick within this guideline you will be fine but that doesn't necessarily mean deviations would result in "biases".
"However, SYBR® Green amplification of larger fragments did not lead to earlier detection as expected in theory".
http://www.pressesagro.be/base/text/v21n1/3.pdf
Also consider are result with the same Ct but not RFU. What would that mean? See below figure right panel.
https://www.researchgate.net/profile/Kahiu_Ngugi/publication/275032499/figure/fig7/AS:294634464006152@1447257739012/Figure-7-Real-Time-PCR-amplification-curves-of-crop-specific-loci-SB3420-and-SB1764.png
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