Quantitative PCR (qPCR) primer design is a critical step when setting up your qPCR or reverse transcription-qPCR assay (RT-qPCR) for gene expression analysis. qPCR primers that anneal poorly or that anneal to more than one sequence during amplification can significantly impact the quality and reliability of your results.
Also, if you are performing a one-step reverse transcription PCR (RT-qPCR), the reverse transcriptase will use the reverse primer to prime the transcription reaction. In this scenario, a poor primer would result in both inefficient reverse transcription and inefficient amplification – a lose–losesituation.
Considering the above, it’s well worth spending the time necessary to design good-quality qPCR primers. This article will tell you exactly how to do that!
The good news is that primers are cheap, so you can easily test several different pairs to choose the best ones for your experiment.
The bad news is that primer testing requires time and patience, so the sooner you get a pair of primers working, the better.
TheNCBI tool Primer-BLASTis widely used for qPCR primer design. There are many other primer design tools available online, including primer3, and PCR suppliers often offer their own design programsfree of charge.
Below arethe main steps involved in qPCR primer design using the NCBI tool Primer-BLAST.
The design steps will be similar if you use other primer design programs, and the information below should give you an idea of the parameters to watch out for.
qPCR Primer Design: Getting Started
The first step in designing primers is to get the nucleotide sequence of your gene of interest.
Go to the Pubmed gene database and search for your gene of interest. You can then filter by species in the right-hand corner of the next screen.
Click on your gene of interest and scroll down until you find the NCBI Reference Sequence (RefSeq) for your gene (e.g. “NM_203483”). Note that there may be multiple sequences if your gene has different isoforms – make sure you click on the isoform you are interested in.
Click on the name and on the next screen, you will see a link to “Pick primers” in the right-hand corner of the screen underneath “Analyze this sequence”. Click on this link to take you to the Primer-BLAST tool.
Primer-Blast Tool
The Primer-BLAST tool has many parameters and options to set. The next sections take you through each of the options in the tool and explains what to set for each one and why.
Parameters for qPCR Primers
This section covers some of the basic settings for your primers, including the PCR product size and melting temperature. There is also an option to include sequences for reverse or forward primers.
Set the following primer parameters:
- PCR product/amplicon size: For efficient amplification, design the primers so that the amplicon is between 70 and 200 bp long.
- Number of primers to return: This is up to you, depending on how many options you want to choose from. It won’t take long for the program to design 10 primer pairs, and this should give you a reasonable chance of finding a suitable pair.
- Melting temperature: As a rule, aim for a minimum of 60°C and a maximum of 63°C; the ideal primer melting temperature is 60°C (with a maximum difference of 3°C in the melting temperatures, Tm, of the two primers). You can use a Tm calculator to determine these temperatures.
Exon/Intron Selection
To avoid amplification of contaminating genomic DNA, design primers so that one-half of the primer hybridizes to the 3′ end of one exon, and the other half to the 5′ end of the adjacent exon.
To do this, simply select “Primer must span an exon–exonjunction.” You don’t need to change the other settings.
Primer pair specificity checking parameters
Use the default settings. The program will use the RefSeq mRNA sequence from the organism you selected to designthe primers.
Checking the Output Screen
Once all the parameters are set, clicking on ‘Get primers’ returns a list of potential forward and reverse primer sequences to choose from. This can take a while but the screen updates periodically to show the time since submission.
Take a look at the options the program returned and pay special attention to the following:
- Make sure the 3′ end of the primer contains a C or G residue because T and A residues bind more easily to DNA in a non-specific way.
- Aim for a GC content of around 40–60% to ensure maximum product stability.
- Avoid self-complementarity to decrease the possibility of primer–dimerformation. Ideally, the primer should have a near-random mix of nucleotides.
Now, pick the best two or three primers, order and test them. Good luck!
If you have any other top tips for qPCR primer design, we’d love to hear from you in the comments!
Need help in other areas of qPCR from setup to data analysis? Check out our top 11 qPCR papers every researcher should know.
Originally published February 6, 2013.Reviewed and republished2017 and April 2021.
Written by New England Biolabs
FAQs
What makes a good primer for qPCR? ›
Both qPCR primers should have a Tm between 59–65oC and be within 1oC of each other (no more than >3oC difference). The Tm also relates to the annealing temperature of the reaction. As a rough rule of thumb, the annealing temperature is approximately 5oC lower than the Tm of the primers.
What are the 3 main strategies for primer design? ›There are 3 strategies for primer design: 1) insert-specific primers, 2) backbone-specific primers, and 3) orientation-specific primers.
How do you design a primer for PCR example? ›- Aim for the GC content to be between 40 and 60% with the 3' of a primer ending in G or C to promote binding. ...
- A good length for PCR primers is generally around 18-30 bases. ...
- Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other.
How to Optimize qPCR using SYBR Green Assays - Ask TaqMan #38
How much primer do I need for qPCR? ›A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM.
What is the difference between qPCR primers and PCR primers? ›The PCR is based on the principle of DNA amplification by primer annealing using Different temperature zones. On the other side, the qPCR relies on the principle of the use of fluorescence probes or dyes that emit fluorescence during the amplification.
How many primers are used in qPCR? ›A. A qPCR assay targeting fungal DNA was used with two sets of forward and reverse primers, which differ mainly at their 3′-ends. The PCR amplicon has no secondary structure issues at the primer binding sites.
How do you know if a primer is good? ›- whether or not your primer pairs are unique, they won't bind to other locations in the genome except your intended gene or DNA fragment.
- will primer pair bind to each other (forming primer dimer)-- (1) self-dimer or (2) hetero-dimer.
Two-step RT-qPCR
commonly poly(A)+ RNA, is used as the starting material and cDNA is produced by elongation from oligo-dT primers, random primers, a blend of oligo-dT primers/random primers, or gene-specific primers using a reverse transcriptase enzyme. An aliquot of this reaction is then added to the qPCR.
The optimal length of primers is generally accepted as 18–24 bp in length. Longer primers will take longer to hybridize, longer to extend, and longer to remove thus produces less amplicon.
What are the three parts of a primer? ›
The primer contains a mixture of substances that perform three basic functions: an initiator, which is an explosive that starts the process when the firing pin hits the primer; a sensitizer, which helps in the ignition process; and a fuel, which sustains the flame and ensures adequate time to light the powder.
Do primers have to be the same length? ›Popular Answers (1) You can use primer set having different lengths. No problem.
What are the 5 key basic reagents used in PCR? ›In general, a complete PCR reaction requires five basic PCR reagents; DNA/RNA template, DNA polymerase, primers (forward and reverse), deoxynucleotide triphosphates (dNTPs) and PCR buffers.
Why do we design primers? ›The primer design is an important step to get an optimal PCR. If you pick up primers without design, the amplification may not work or give you "strange" results, for example if the primer can hybridize at another position in the genome.
Why do we use 2 primers in a PCR reaction? ›Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied.
How do you find the primer sequence? ›Sequence Viewer: Using Primer-BLAST - YouTube
How do you optimize primer concentration for qPCR? ›Optimization of qPCR Conditions
One approach to optimizing primer concentrations is to create a matrix of reactions. This is used to test a range of concentrations for each primer against different concentrations of the partner primer.
SYBR Green qPCR - YouTube
How do you choose annealing temperature for qPCR? ›When performing qPCR it is ideal to have your probe Tm about 5-10 degrees higher than your primer Tms. The annealing temperature should be set 3-5 degrees lower than the lowest primer Tm. Use this as a general guideline, but note that optimization may still be necessary.
What happens if you add too much primer to a PCR? ›Too much primer was added
Using a high concentration of primers may increase the chance of primers binding to nonspecific sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.
What concentration of primer should I use for PCR? ›
In setting up PCR, primers are added to the reaction in the range of 0.1–1 μM. For primers with degenerate bases or those used in long PCR, primer concentrations of 0.3–1 μM are often favorable.
Does primer concentration affect qPCR? ›The observed Cq differences for a given RT-qPCR assay are due to the varying primer concentrations, as these were the only variable reaction parameters. The performance of the majority of the RT-qPCR assays were significantly dependent on primer concentration (Fig.
Can I use same primers for PCR and qPCR? ›The primers that work on qPCR, can actually work on endpoind PCR. We test the primers for qPCR by performing gradient-endpoint PCR before. Just keep in mind that you must use the same type of template (cDNA or DNA) in each case.
Can you use regular primers for qPCR? ›for the conventional/ semi-q PCR you can use the same primers in fact it is better to first standardize the annealing temperature of your primers with semi-q PCR before going for Real-time it saves you plenty of reagents! All the best!
How many primers do I need for RT-PCR? ›RT-PCR amplification of a particular RNA sequence requires two PCR primers that are specific for the gene transcript of interest. The primer design should allow differentiation between the amplified product from cDNA and an amplified product derived from contaminating genomic DNA.
How do you choose forward and reverse primers? ›Forward and reverse primers explained - YouTube
How do I verify qPCR primers? ›Spin the plate for 2 minutes at 1200rpm and then run the qPCR reaction. The melt-curve performed at the end of the qPCR cycles is important in confirming the specificity of the primer annealing. The curve should display a single peak with no shouldering (see below).
What is a good primer efficiency? ›You want to achieve primer efficiencies between 90 and 110%. If your primers are not within that range it is most likely an error with this qPCR reaction.
Why is GC content important in primers? ›GC bonds contribute more to the stability—i.e., increased melting temperatures—of primer and template, binding more than AT bonds. Primers with 40% to 60% GC content ensure stable binding of primer and template.
How much RNA do you need for qPCR? ›For initial experiments, we recommend using between 0.5 to 1 µg of input total RNA, and using 1 µl of either undiluted template or template pre-diluted 1:10 for each 25-µl RT² qPCR Assay reaction.
How many types of primers are there? ›
Types of Primers. There are three basic types of primers: oil-based, latex and pigmented shellac primer. Each has its strengths and weaknesses and works best on certain surfaces and in particular circumstances.
What 2 types of primers are used in modern cartridges? ›Berdan and Boxer primers are two different types of centerfire primers used in modern metallic cartridge ammunition. The main difference between the two is that Boxer primers have a self-contained anvil within the primer whereas Berdan primers have an anvil that is integral to the primer pocket on the cartridge case.
What would happen if the primers are incorrect? ›An incorrect PCR primer can lead to a failed reaction- one in which the wrong gene fragment or no fragment is synthesized. Careful construction or selection of the primer sequence set for your PCR experiments will result in uncontaminated and accurate genetic synthesis.
Why is mgcl2 used in PCR? ›MgCl2 (Magnesium chloride) is an essential ingredient of the PCR master mix. Acting as a cofactor, it enhances the enzymatic activity of DNA polymerase, thereby boosting DNA amplification.
What are four important PCR applications? ›We present a survey of the following applications of PCR: 1) The amplification of gene fragments as fast alternative of cloning. 2) The modification of DNA fragments. 3) The sensitive detection of pathogenic microorganisms, if desired followed by an accurate genotyping. 4) DNA analysis of arachaeological specimens.
Why is a PCR cycle repeated 30 times? ›At the end of a cycle of these three steps, each target region of DNA in the vial has been duplicated. This cycle is usually repeated 30 times. Each new DNA piece can act in the next cycle as a new template, so after 30 cycles, 1 million copies of a single fragment of DNA can be produced (Scheme - Diagram of PCR).
How do I design qPCR primers NCBI? ›NCBI Primer BLAST | In silico PCR primer designing and validation
How much primer do I need for qPCR? ›A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a titration of primer concentrations between 100 and 500 nM.
What type of primers are used in qPCR? ›Two-step RT-qPCR
commonly poly(A)+ RNA, is used as the starting material and cDNA is produced by elongation from oligo-dT primers, random primers, a blend of oligo-dT primers/random primers, or gene-specific primers using a reverse transcriptase enzyme. An aliquot of this reaction is then added to the qPCR.
A. A qPCR assay targeting fungal DNA was used with two sets of forward and reverse primers, which differ mainly at their 3′-ends. The PCR amplicon has no secondary structure issues at the primer binding sites.
How long should qPCR primers be? ›
The optimal length of primers is generally accepted as 18–24 bp in length. Longer primers will take longer to hybridize, longer to extend, and longer to remove thus produces less amplicon.
Can you use the same primers for PCR and qPCR? ›The primers that work on qPCR, can actually work on endpoind PCR. We test the primers for qPCR by performing gradient-endpoint PCR before. Just keep in mind that you must use the same type of template (cDNA or DNA) in each case.
What happens if you add too much primer to a PCR? ›Too much primer was added
Using a high concentration of primers may increase the chance of primers binding to nonspecific sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.
In setting up PCR, primers are added to the reaction in the range of 0.1–1 μM. For primers with degenerate bases or those used in long PCR, primer concentrations of 0.3–1 μM are often favorable.
What is the difference between qPCR primers and PCR primers? ›The PCR is based on the principle of DNA amplification by primer annealing using Different temperature zones. On the other side, the qPCR relies on the principle of the use of fluorescence probes or dyes that emit fluorescence during the amplification.
Can you use regular primers for qPCR? ›for the conventional/ semi-q PCR you can use the same primers in fact it is better to first standardize the annealing temperature of your primers with semi-q PCR before going for Real-time it saves you plenty of reagents! All the best!
How much RNA do you need for qPCR? ›For initial experiments, we recommend using between 0.5 to 1 µg of input total RNA, and using 1 µl of either undiluted template or template pre-diluted 1:10 for each 25-µl RT² qPCR Assay reaction.
How do you choose forward and reverse primers? ›Forward and reverse primers explained - YouTube