Hi Kais, as by previous excellent answers you are already well aware to the fact that for a maximum qPCR efficiency amplicons are kept smaller. The problem with longer amplicons raises when lets say you have less specific primers (which is a frequent, very common and quite disturbing issue anyhow known as primer designing) so here the longer amplicons may give you more than 1 melt peaks as they may dissociate more than 1 time due to possible secondary structures.

Also an increased length of amplicon no doubt will increase the chances of detection something not desired, which may entirely lie outside to your GOI or target.

Hence for Sybr Green assay its very important to check your primers efficiency first regardless of the amplicon length status and select the best set of primers.

Also there is an intrinsic creepy nature of Sybr Green which is its "redistribution" just after its release from the melted part of duplex during the melting phase but again into the same duplex adjacently, which is still hanging in its double stranded form and not opened yet fully as its Tm not reached. So in case of quite longer amplicons, you can say that the amount of fluorescence of SG detected by the machine is not in fact 100% corresponds to the amount of actual product or amplicon formed in the reaction. So longer amplicons, multiple issues & this is the fact, avoid it if you can.

Dear Kais,

longer amplicons decrease the efficiency of a qPCR. To be robust and comparable, the efficiency of  a qPCR reaction should be between 90% and 110%.

You can always run qPCR designed for longer products, but you will need to adjust cycling conditions and do more quality assurance and troubleshooting to be confident in CT values. Also, I would tend to use SYBR green for that matter vs. probe assay on a large amplicon. 

Nicolas

 Thanks Dr. Nicolas Tremblay

• I have used amplicons up to 300bp with acceptable efficiency 
• However it is always better to keep between 100-200bp for consistent efficiency >85%
• I have on the odd occasion tried products at 400bp (by virtue of necessity) and efficiency ~75% which is OK for efficiency corrected relative expression (pfaffl method) but this magnitude of efficiency is not ideal 
• Thus 100-200bp ideal; 200-300 acceptable but not optimal; > 300bp not recommended 

Thanks Dear Laurence Stuart Hall, 

I think i need to adjust cycling conditions for longer products for acceptable efficiency,

Best regards 

Hi Kais

• yes longer extension times Will definitely help for products >200bp
• In addition for these larger products addition of 5% DMSO to your qPCR reactions can also enhance efficiency: 
• In a 20ul reaction add 10ul of your 2x sybr green master mix and 1ul of each primer @2.5uM = 2.5 pMoles/ul = 2.5 pMoles & 1ul of DMSO
• Finally for amplicons > 200bp sometimes increasing the Mg concentration by 1mM can boost efficiency by increased by increasing Taq processivity 

 Hi Kais, as by previous excellent answers you are already well aware to the fact that for a maximum qPCR efficiency amplicons are kept smaller. The problem with longer amplicons raises when lets say you have less specific primers (which is a frequent, very common and quite disturbing issue anyhow known as primer designing) so here the longer amplicons may give you more than 1 melt peaks as they may dissociate more than 1 time due to possible secondary structures.

Also an increased length of amplicon no doubt will increase the chances of detection something not desired, which may entirely lie outside to your GOI or target.

Hence for Sybr Green assay its very important to check your primers efficiency first regardless of the amplicon length status and select the best set of primers.

Also there is an intrinsic creepy nature of Sybr Green which is its "redistribution" just after its release from the melted part of duplex during the melting phase but again into the same duplex adjacently, which is still hanging in its double stranded form and not opened yet fully as its Tm not reached. So in case of quite longer amplicons, you can say that the amount of fluorescence of SG detected by the machine is not in fact 100% corresponds to the amount of actual product or amplicon formed in the reaction. So longer amplicons, multiple issues & this is the fact, avoid it if you can.

 Dear Jawwad Ahmad

Thanks for the detailed explanation,

Best regards 

• I agree to an extent Jawwad
• Namely that larger amplicons increase the probability of secondary peaks
• However in the main secondary peaks; both primer diners and non specific primers are due to non optimal primers and sybr green qPCR conditions; in particular stringent annealing temp to provide maximal specificity
• To Andrews these 3 points 

• https://www.idtdna.com/calc/analyzer
• https://www.idtdna.com/Primerquest/Home/Index

• https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

Dear Kais:

Firstly I thank you for this wonderful discussion. I just want to add some thing:

The amplicons in real time PCR designed to be as small as possible because:

1. We need it to be very specific to avoid multi-peaks according to un-wanted annealing cases.

2. The result that we need is the Ct value not the big amplicon size as we do not need run the product on gel elctrophoreces i. e. we don't need to view bands as the measurement of results depends on Ct (Cycle of threshold) to calculate even quantity or gene expression.

3. Efficiency is just a parameter to give us indication about accuracy of our reaction.

• I cannot comment specifically on the last speaker but amplicons in qPCR are made small to ensure high PCR efficiency 
• Efficiencies of > 85% are imperative to ensure you can accurately calculate relative gene expression based on the delta delta Ct or Livak method
• This is because the Livak method in its calculations of changes in gene expression assumes a perfect doubling rate of '2' so to be valid the actual situation must approximate to this (>0.85 x2)
• http://www.gene-quantification.net/livak-2001.pdf
• If the actual efficiency verified by standard curve based on serial dilution of your cDNA is ~ 75% then a 'correction coefficient' should be included in the calculation by using the so called Pfaffl method: 
• http://www.gene-quantification.com/chapter-3-pfaffl.pdf
• http://www.bio-rad.com/en-uk/applications-technologies/real-time-pcr-experimental-design
• Smaller amplicons are not in any substantial way linked to non specific amplicons: this is a function of primer design and specificity as detailed in my last answer

Dear Laurence Stuart Hall,

Thank you very much for the useful references 

Best regards