Hi Ashish,

did you check your primer efficiencies for both primer pairs? This could be one reason for different CT values. Furthermore, did you run your samples in one real-time PCR together? You might want to keep in mind, that if you increase elongation time because of one longer product in the same time there will be higher amplification of your smaller product since it is more than two times smaller. This would lead to a much smaller CT value for your smaller product.

Try running them in separate PCRs (include inter-run calibration in both PCRs like a 'house keeping gene' with similar amplicon size as your marker), adjust primer efficiency and again compare your CT-values.

Hope this helps!

Cheers Hilke

Hi Ashish,

yes, amplicon sizes has a real impact on PCR efficiency and for qPCR it is well recommended for the size to be around 100-150bP.

just imagine with same primer efficiency and same conditions (mix, template and Tm), for 137 bp and 343 bp, for which of these ones will the Taq be efficient since lot of PCR programs take into account length for elongation time.

fred

Hi Ashish,

Is it a SYBR green RT?

If so, you need to consider the logic of having a relation between the rfu and the amplicon size, as larger size should theoretically influenced by more SYBR molecules as therefore emit and record higher rfu.

Unspecific bands also might influence your rfu, melting curve should give you a great idea about what is really happening!

The huge differences in expression values is a very generalized statement, if you please give some details, is the observation based on the delta-CT or the deltadelta-CT, or the fold change?

What about your internal and biological control? Your controls should have normalized the differences you have, otherwise the previous comments should help in improving your CT values.

Best wishes

Hi

For me the best way is to leave only one primer set with lower cycle number. Of course, usually I do sequencing of amplicon with "Sanger" to see target sequence and avoid unspecific region amplification even in the case of TaqMan.

Primer efficiency is somewhat affected by amplicon size. Ideally, you want your amplicons 100-150bp long. But that doesn't mean you can't get efficient amplification of larger products. Ct value is affected by both primer efficiency and relative gene expression.

What do your standard curves look like? If the calculated efficiencies are 95-105% for your primers, then you can use them.

The difference in relative fluorescence will affect the height of the RFU plot, but not the slope of the lines. So, the difference in product size regarding RFU shouldn't matter.

After getting realtime results I ran the product on 1 per cent agarose gel. The marker which has larger product size gave smear like appearance whereas the other marker which has smaller amplicon gave clear and good bands. I have attached the ppt here, please find the attachment.

Actually we are not keeping extension phase here in real-time, after initial denaturation (2 min) there is denaturation phase (15 sec), then annealing phase (30 sec), and then it is coming back to denaturation phase-annealing phase. This is how 39 cycles have been completed. So in my conclusion the 30 sec of annealing/extension was not sufficient for proper formation of product, and in attached ppt there is appearance of smear instead of bands which indicates all products are not of equal length.

As others have pointed out, yes, amplicon size can affect your Ct values so comparing Ct's from two markers with very different sizes is not the best practice. Here's another suggestion... if you want to try and make direct comparisons between your two markers without re-designing your primers, you can normalize and directly compare them by converting the Ct values to estimated starting template amounts using a formula first derived by Alvarez et al. (2007) Model based analysis of real-time PCR data from DNA binding dye protocols DOI: 10.1186/1471-2105-8-85. The formula includes a variable to account for amplicon size. My group used a variation of this formula for just this purpose in Grimes et al (2014) DOI:10.1186/1471-2164-15-31