qPCR primer design with NCBI Primer-BLAST

Learn the critical parameters for efficient qPCR primer design, including optimal PCR product length (70-200 bp), melting temperature range (60-63°C), and the importance of spanning exon-exon junctions to avoid amplifying genomic DNA.

Navigate to the NCBI website and search for your gene of interest (e.g., ACE2). Locate the NCBI reference sequence for the correct isoform and click on the gene name to access the sequence details.

Select 'Pick primers' from the right-hand side of the sequence page to open the Primer-BLAST interface for designing primers against your selected sequence.

Set the parameters: change maximum number of primers to 200, set melting temperature to 60°C, and enable the option to span exon-exon junctions. Use default settings for primer pair specificity to search against RefSeq mRNA sequences.

Click 'Get primers' and then submit to run the analysis. Review the output page showing primer pair locations on the gene graphic and the list of candidate primer pairs.

Examine each primer pair's sequence, length, melting temperature, GC content (40-60%), self-complementarity, expected product length, and specificity to ensure they meet design criteria and bind only to your target gene.

Compare all returned primer pairs and select the best two to three options based on their specifications and specificity. Order the selected primers and proceed to experimental validation.