Below is a comprehensive guide detailing the step-by-step process of designing qPCR primers from scratch using NCBI Primer BLAST, followed by thorough double-checking to ensure primer specificity and reliability.
1. Accession number retrieval: Obtain the accession number of your gene of interest. One valuable source to find this information if the NCBI nucleotide database: https://www.ncbi.nlm.nih.gov/nucleotide/. NM accession number links to the mRNA record in the Nucleotide database. Make sure you select the right gene by organism. For example: NM_009507.4von Hippel Lindau tumor suppressor gene in Mus musculus
2. Go to the NCBI website (https://www.ncbi.nlm.nih.gov/) and navigate to the "Primer BLAST" tool under the "Tools" menu or directly visit the Primer BLAST page (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (1)
a. Enter the accession number (example: NM_009507.4; should always start with “NM_”).
b. Set primer parameters: use all the default settings, except, in the “exon junction span” pull-down menu, select “Primer must span an exon-exon junction”.
· This ensures the primer pair will read cDNA only.
c. Also, for “PCR product length”, change the minimum from the default of 70 to 50, and the maximum from the default of 1000 to 200 (final range 50-200).
d. Make sure the species is set to Mus musculus (or your organism of choice like Homo sapiens)
e. Click the big blue “Get Primers” button at the bottom to initiate the primer design process
f. It will take some time to process, at least 30 seconds and sometimes up to 5 minutes.
3. Finally it will display a list of ten primer pairs, with a graphical representation of the exons of the gene and where the primers match to.
4. Double-check primer specificity: Choose any one of the primer pairs, making sure it doesn’t have off-target matches (which would show up on the “products on potentially unintended templates” see image below)
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5. Record details: the primer sequence, length, melting temperature, GC content, and amplicon length for each primer (forward and reverse primers listed separately)
6. Primers should fall under the following requirements:
a. length 18-21bp
b. GC content 50% ± 10%
c. melting temperature 60° ±3°
d. no homo- or heterodimers
7. To further evaluate these parameters, use the IDT Oligo Analyser tool: https://www.idtdna.com/calc/analyzer. (2)
a. Enter your sequence and click the orange “Analyze” button, and ensure that the melting temperature, GC content, and primer length are within bounds.
b. Once those requirements are fulfilled, check for hairpin loop structures with the “hairpin” button.
· Ensure that the melting temperature of the hairpin structures is lower than the annealing temperature of the PCR reaction (50°C).
c. Next, check for self-dimerisationevents with the “Self-Dimer” button.
· Make sure the ΔG of the homodimers are greater than -9 kcal/mole (so, less negative values or positive values).
d. Check for heterodimers (> -9 kcal/mol), checking the forward and reverse primers against each other.
e. This tool allows you to move to ordering if the sequences work for you.
8. Optionally, you can check the primer pair using UCSC’s in silico PCR tool at http://genome.ucsc.edu/cgi-bin/hgPcr (3) to make sure your primer pair actually amplifies something.
a. Ignore what it says for amplicon length, since the in silico tool will use genomic DNA and thus insert a huge intron into your amplicon.
b. Sometimes the in silico tool won’t give you a result, but that doesn’t necessarily mean your primer pair doesn’t work.
9. Order primers: If a primer pair passes the IDT check and UCSC’s in silico tool, you can proceed to ordering.
a. It is recommended that the primers details are triple checked, preferably by another person.
10. Experimental validation: Before using the primers in your experiments, validate their functionality through PCR or qPCR assays using appropriate controls and conditions.You can verify in an agarose gel that the product size matches the expected.