TITLE: qPCR Primer Design
AUTHORS: Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for designing qPCR primers for analysis of transcript abundance using SYBR Green chemistry.</div><div class = "text-block"><span>There are many good guides about how to do this online such as </span><a href="https://www.bio-rad.com/en-us/applications-technologies/qpcr-assay-design-optimization?ID=LUSO7RIVK" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">this</span></a><span>, from which the following protocol is derived.</span></div><div class = "text-block">Reference for mfold/UNAfold</div><div class = "text-block"><span>https://doi.org/</span><span style = "font-style:italic;">10.1093</span><span style = "font-style:italic;font-style:italic;">/</span><span style = "font-style:italic;">nar</span><span style = "font-style:italic;font-style:italic;">/</span><span style = "font-style:italic;">gkg595</span></div></div>

[STEPS]
?. [Identify target sequence]
Decide on a gene of interest, download the transcript sequence in .gb format and upload to a new folder on Benchling for analysis.
If analyzing an endogenous (as compared to transgene), gene sequences can be obtained from public databases such as NCBI. For many plant genes, my preference is to use Phytozome, but you should choose whichever database has the most recent and accurate transcriptome models.One thing to be aware of is that genes can have multiple splice forms. You may, or may not wish to perform splice form specific transcript quantification. In most instances, you are likely interested in "gene expression", or the sum of transcripts originating from a gene. In this case, perform an alignment of known splice form sequences using clustal omega and retain only the parts which are common to all.
?. [Use Primer-BLAST to test for specificity]
Navigate to NCBI Primer-BLAST in your browser
?. [Use Primer-BLAST to test for specificity]
Adjust parameters to suit qPCR assays and record the search conditions in your Lab book.Amplicon size to 70-150 bpMinimum of 4 mismatchesRefSeq mRNA databaseUnder the organism tab, enter the name of the species of interest e.g. Solanum tuberosum
You can also include information about intron-exon boundaries, however, this is often too stringent and will yield no positive results.
?. [Use Primer-BLAST to test for specificity]
Paste in the transcript sequence against which you want to design primers (from step 1) and click "Get Primers".
?. [Use Primer-BLAST to test for specificity]
From the results choose primers which have a single perfect match using BLAST (this should be against record the following details in a table for the purpose of reporting according to the format above:Primer forward sequencePrimer reverse sequenceAmplicon size
?. [Check primer properties]
Open IDT's OligoAnalyzer Tool in a new tab for QC analysis of primer properties.
?. [Check primer properties]
Change parameter settings to "qPCR" on the dropdown menu on the right and adjust concentration settings: [Oligo] = 0.33 nM, [Na+] = 50 mM, [Mg2+] = 3 mM, [dNTP] = 1.2 mM
Note these parameter settings are adapted for use with SSoAdvanced Universal SYBR Green Supermix. The precise parameters will vary depending on the buffer components in the PCR reaction mix being used.
?. [Check primer properties]
Enter the first primer sequence from Primer-BLAST and run
?. [Check primer properties]
Analyze hairpin formation and record the highest Tm. Record the results in the table.
Tm value should be lower than the annealing temperature of the primers (e.g. 60 oC if that is what the assay was designed at). Otherwise, primer sequence may be prone to forming hairpin structures under assay conditions resulting in inaccurate results.
?. [Check primer properties]
Analyze self-dimer free energy and record thelowest dG in a table. Record the results in the table.
dG value should be > -9. Anything lower than this fails the quality control check as the sequence has too high a chance of forming primer-dimer amplification products leading to inaccurate results.
?. [Check primer properties]
Analyze heterodimer free energy by entering the reverse primer as a secondary sequence.  Record the results in the table.
dG value should be > -9. Anything lower than this fails the quality control check as the sequence has too high a chance of forming primer-dimer amplification products leading to inaccurate results.
?. [Check primer properties]
If the forward primer passes the QC checks in steps 9-11, repeat for the reverse primer.
In the case that the primer sequence fails QC, return to the Primer-BLAST results and select another pair of primers for analysis. It is common to need to do this, in most instances, it should be possible to find suitable primers.
?. [Check amplicon properties]
Open the software UNAfold in a new tab on your browser to test for secondary structure formation by the amplicon sequence to be amplified by primers.
Ideally, your primers should pass this QC as well, but in the event, it is not possible to find a pair that passes both the primer and amplicon QC it is worth progressing to test experimentally.
?. [Check amplicon properties]
Set UNAfold parameters for analysis of assay amplicon:Chose to fold DNASet the concentration of Mg++ to 3 mM
?. [Check amplicon properties]
Run UNAfold
?. [Check amplicon properties]
Inspect the predicted structures formed by the amplicon.
The highest Tm for any of the predicted structures should be less than the annealing temperature of the primers (e.g. >60 oC if that is what they were designed at).