TITLE: qPCR Primer Design
AUTHORS: Steven J Burgess

[DESCRIPTION]
Protocol for designing qPCR primers for analysis of transcript abundance using SYBR Green chemistry.

There are many good guides about how to do this online such as this, from which the following protocol is derived.

Reference for mfold/UNAfold
https://doi.org/10.1093/nar/gkg595


[GUIDELINES]
Preferred properties of qPCR primers:
(Note: These guidelines represent the ideal situation which may not always be achievable)
each primer should be between 15–30 bp in length
theoretical Tm of the two primers should be within 2°C of each other.
Try to avoid G/C clamps at the 3´ ends of the primers to prevent these oligos from folding on themselves or annealing nonspecifically.
The five bases at the 5´ terminal end generally should contain no more than two guanines and cytosines, although it is acceptable to have three in the final 5 bases if no two are adjacent.
If possible have dA nucleotides near the 3' end to allow for UNG activity to prevent primer-dimers
Since thymidine tends to mis- prime more readily than the other bases, a 3´ terminal T should be avoided if possible.
The 5´ end of the primers should not contain an inverted repeat sequence that would allow it to fold on itself.

[STEPS]
SECTION: Identify target sequence
1. Decide on a gene of interest, download the transcript sequence in .gb format and upload to a new folder on Benchling for analysis.

 
 
SECTION: Use Primer-BLAST to test for specificity
2. Navigate to NCBI Primer-BLAST in your browser
SECTION: Use Primer-BLAST to test for specificity
3. Adjust parameters to suit qPCR assays and record the search conditions in your Lab book.
Amplicon size to 70-150 bp
Minimum of 4 mismatches
RefSeq mRNA database
Under the organism tab, enter the name of the species of interest e.g. Solanum tuberosum
 

SECTION: Use Primer-BLAST to test for specificity
4. Paste in the transcript sequence against which you want to design primers (from step 1) and click "Get Primers".

SECTION: Use Primer-BLAST to test for specificity
5. 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 sequence
Primer reverse sequence
Amplicon size

SECTION: Check primer properties
6. Open IDT's OligoAnalyzer Tool in a new tab for QC analysis of primer properties.
SECTION: Check primer properties
7. 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
 


SECTION: Check primer properties
8. Enter the first primer sequence from Primer-BLAST and run
SECTION: Check primer properties
9. Analyze hairpin formation and record the highest Tm. Record the results in the table.
 


SECTION: Check primer properties
10. Analyze self-dimer free energy and record thelowest dG in a table. Record the results in the table.
 

SECTION: Check primer properties
11. Analyze heterodimer free energy by entering the reverse primer as a secondary sequence.  Record the results in the table.
 

SECTION: Check primer properties
12. If the forward primer passes the QC checks in steps 9-11, repeat for the reverse primer. 
 

SECTION: Check amplicon properties
13. 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.


 

SECTION: Check amplicon properties
14. Set UNAfold parameters for analysis of assay amplicon:
Chose to fold DNA
Set the concentration of Mg++ to 3 mM

SECTION: Check amplicon properties
15. Run UNAfold
SECTION: Check amplicon properties
16. Inspect the predicted structures formed by the amplicon.