I'm using Invitrogen superscript II and SsoAdvanced SYBR green for qPCR (pcrs work but I'm not sure if these systems are completely compatible). Annealing is at 63 and shows no primer dimers at this temperature on acrylamide gel (only some show primer dimers at lower annealing temp ~57 degrees)
The weird thing about this increase in melt curve background is that it doesn't seem to change in a gradient annealing program, whereas samples that have primer dimers increase with lower temperatures.
Here's the melt curve for one of my primer sets that has a clean band by electrophoresis:
Hi there
as a veteran of qPCR I would not worry about the shoulder on your curve, especially if the data you intend to generate is relative expression and not absolute copy number and especially since your product looks clean by agarose gel electrophoresis. I have generated alot of good relative gene expression data from melt profiles that look like that. Occasionally I have that primer pairs with shoulders potentially indicating primer dimers disappear if you use a lower concentration of cDNA and especially lower primer concentration: To be systematic you could try Varying your respective primer concentrations from 50nM to 300nM as shown on page 26 of the attached pdf qPCR over viewYou could also try adding 2% DMSO to your qPCR reaction if you are concerned. In my experience the most pertinent thing is signal to noise and the fact that your specific peak generates a much higher signal than the accompanying shoulder is the most pertinent practical consideration. Moreover as already mentioned the delta G of any putative dimer structure is < 5kcal/mol which indicates poor thermodynamic stability and thus any such structure is likely not to exist in any meaningful steady state way
When I perform qPCR expecially in the context of relative expression data I take the pargamatic approach from years of experience that if PCR looks clean by gel electrophoresis then the signal to noise is likely to be good enough to generate good qPCR data all other things being equal. As mentioned you could try a cDNA primer titration to try and reduce or eliminate the shoulder but I wouldnt worry too much if that has no effect given the strong specific signal on your melt curve attached and the fact that your PCR looks clean by gel. If you data is relative gene expression then any potential effect of this shoulder could well be masked by sample variance and in any case with high signal to noise any such subversion will have no effect on the actual gene expression trend which is really what matters in these sorts of experiments
If however you do decide to go ahead and redesgn primers find attached a link to a primer design document prepared by me and resident in my 'qPCR' drop box folder: Apart from talking about the fact primers ending in 2 or 3 G/Cs are contraindicated see the IDT primer design tool which will allow you to calculate the free energy (delta G) of any such structure and thus the likely hood in reality that it will exist in real time in a significant steady state amount and thus pose a tangible rsik toundermining amplification efficiency in your qPCR reaction
For more information on primer design see an archived answer of mine pertaining to this question:
in essence however you need to1. Make sure your primer Tms are no more than 2C Apart and also ensure that the Tm of both primers is 55C to 65c: To calculate your primer Tms there are lots of programs on line or links to such programs, e.g primer 3 oligo calculator or IDT primer design tool alternatively just google those terms. When computing Tms for accuracy be sure to include your primer concentrations. These should incidentally be about 1pmole/ul in your final reaction. That is 2ul of primers at 10uM (= 10 pmoles/ul) in a 20ul reaction; your magnesium conc which is usually 1.5-2mM and your final dNTP conc.See your data sheet that comes with your taq for specifics 2. In terms of actual base composition this will mean both primers should be 18bp to 21bp in length and have a GC content approximating to 50% to 60%3. Beyond this pay particular attention to the last 5bp of your primer at 3' end which principally determine efficiency of priming and to a less extent specificity: if possible try and ensure that the last 5bp contain 3 G/C residues for efficient hybridisation although try not to end the primer in either GG CC or GC residues. Rather GT GA CT or CA would be preferable4. Also try to avoid runs of the same bases any where within the primer if at all possible 5. Having done the above finally you need to screen primers from intra complimentarity that is binding within themselves via haiir pin loops and inter complimentarity; that is binding to each other resulting in primer dimersI have the 2 following primers with extremely far apart Tm.
https://www.researchgate.net/post/I_have_the_2_following_primers_with_extremely_far_apart_Tm_Could_someone_advise_on_the_protocol_for_the_PCR_reaction_Thanks?_tpcectx=profile_answers [accessed Jun 2, 2015].Do you have a detailed RT-PCR protocol?. Available from: https://www.researchgate.net/post/Do_you_have_a_detailed_RT-PCR_protocol?_tpcectx=profile_answers [accessed Jun 10, 2015].
What else can I try for primer optimization in qPCR?. Available from: https://www.researchgate.net/post/What_else_can_I_try_for_primer_optimization_in_qPCR/1 [accessed Jun 16, 2015].
https://www.dropbox.com/s/xfoig78a15yotkh/Introduction_to_Quantitative_PCR_web.pdf?dl=0
https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0
Is it possible that your target sequence has areas where it may partially self-anneal? This would be unaffected by primer annealing temperature, remain consistent with a single amplification product, and occur regardless of PCR optimization. You could try designing primers in a different region of your target sequence or, depending on the sensitivity you need for your assay, just set your threshold above the background.
I have checked the amplicons in silico and made sure that the delta g of secondary structures are less than - 5 kcal/mole. So I'm still not sure why I'm getting this shoulder in the melt curve.
Hi.
I also know this fact. Some of my melting curves looked the same by using the LoopTag-system or EvaGreen. For my point of view it has something to do with the target sequence/amplicon because if i change the primers the effect may be reduced or enhanced. What is the length of your amplicon and which program you used for in silico analysis?
Hi there
as a veteran of qPCR I would not worry about the shoulder on your curve, especially if the data you intend to generate is relative expression and not absolute copy number and especially since your product looks clean by agarose gel electrophoresis. I have generated alot of good relative gene expression data from melt profiles that look like that. Occasionally I have that primer pairs with shoulders potentially indicating primer dimers disappear if you use a lower concentration of cDNA and especially lower primer concentration: To be systematic you could try Varying your respective primer concentrations from 50nM to 300nM as shown on page 26 of the attached pdf qPCR over viewYou could also try adding 2% DMSO to your qPCR reaction if you are concerned. In my experience the most pertinent thing is signal to noise and the fact that your specific peak generates a much higher signal than the accompanying shoulder is the most pertinent practical consideration. Moreover as already mentioned the delta G of any putative dimer structure is < 5kcal/mol which indicates poor thermodynamic stability and thus any such structure is likely not to exist in any meaningful steady state way
When I perform qPCR expecially in the context of relative expression data I take the pargamatic approach from years of experience that if PCR looks clean by gel electrophoresis then the signal to noise is likely to be good enough to generate good qPCR data all other things being equal. As mentioned you could try a cDNA primer titration to try and reduce or eliminate the shoulder but I wouldnt worry too much if that has no effect given the strong specific signal on your melt curve attached and the fact that your PCR looks clean by gel. If you data is relative gene expression then any potential effect of this shoulder could well be masked by sample variance and in any case with high signal to noise any such subversion will have no effect on the actual gene expression trend which is really what matters in these sorts of experiments
If however you do decide to go ahead and redesgn primers find attached a link to a primer design document prepared by me and resident in my 'qPCR' drop box folder: Apart from talking about the fact primers ending in 2 or 3 G/Cs are contraindicated see the IDT primer design tool which will allow you to calculate the free energy (delta G) of any such structure and thus the likely hood in reality that it will exist in real time in a significant steady state amount and thus pose a tangible rsik toundermining amplification efficiency in your qPCR reaction
For more information on primer design see an archived answer of mine pertaining to this question:
in essence however you need to1. Make sure your primer Tms are no more than 2C Apart and also ensure that the Tm of both primers is 55C to 65c: To calculate your primer Tms there are lots of programs on line or links to such programs, e.g primer 3 oligo calculator or IDT primer design tool alternatively just google those terms. When computing Tms for accuracy be sure to include your primer concentrations. These should incidentally be about 1pmole/ul in your final reaction. That is 2ul of primers at 10uM (= 10 pmoles/ul) in a 20ul reaction; your magnesium conc which is usually 1.5-2mM and your final dNTP conc.See your data sheet that comes with your taq for specifics 2. In terms of actual base composition this will mean both primers should be 18bp to 21bp in length and have a GC content approximating to 50% to 60%3. Beyond this pay particular attention to the last 5bp of your primer at 3' end which principally determine efficiency of priming and to a less extent specificity: if possible try and ensure that the last 5bp contain 3 G/C residues for efficient hybridisation although try not to end the primer in either GG CC or GC residues. Rather GT GA CT or CA would be preferable4. Also try to avoid runs of the same bases any where within the primer if at all possible 5. Having done the above finally you need to screen primers from intra complimentarity that is binding within themselves via haiir pin loops and inter complimentarity; that is binding to each other resulting in primer dimersI have the 2 following primers with extremely far apart Tm.
https://www.researchgate.net/post/I_have_the_2_following_primers_with_extremely_far_apart_Tm_Could_someone_advise_on_the_protocol_for_the_PCR_reaction_Thanks?_tpcectx=profile_answers [accessed Jun 2, 2015].Do you have a detailed RT-PCR protocol?. Available from: https://www.researchgate.net/post/Do_you_have_a_detailed_RT-PCR_protocol?_tpcectx=profile_answers [accessed Jun 10, 2015].
What else can I try for primer optimization in qPCR?. Available from: https://www.researchgate.net/post/What_else_can_I_try_for_primer_optimization_in_qPCR/1 [accessed Jun 16, 2015].
https://www.dropbox.com/s/xfoig78a15yotkh/Introduction_to_Quantitative_PCR_web.pdf?dl=0
https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0
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