The most common qPCR reactions incorporate SYBR Green dye into PCR products. Careful primer design is critical. If primer dimers or other non-specific PCR products form, they will incorporate SYBR Green dye. This may lead to inaccurate quantification, especially when detecting sequences of low abundance. Some important guidelines for designing primers for qPCR experiments follow:

1) Choose primers to give a product (amplicon) of 250-400 bp. SYBR Green dye fluorescence depends on the presence of double-stranded DNA, so products shouldn't be too short. If necessary, primers that generate amplicons as small as 100 bp may also be acceptable. Template regions with obvious secondary structures or long runs of the same nucleotide should be avoided when designing primers. Avoid a 3' terminal T on primers if possible. Thymidine tends to misprime more readily than other bases.

2) Check the Tm. Primers should anneal to DNA template at 60 C. Usually a Tm of 65 oC will be sufficient. This can be checked with the oligo analyzer

3) Check for primer dimers. (The link listed above can be used).

4) If analyzing cDNA, design primers within a region of the genome that is transcribed. Coding sequence can be used, and for some studies, 5' untranslated sequences may be transcribed and can be used for qPCR. For cDNA from eukaryotes, it is also possible to design primers to span exon-exon boundaries. If this approach is used, it is worth noting that these primers may detect some splice variants but not others. For example, the 5' end of the forward primer might be complementary to the end of exon 1, and the 3' end of the forward primer might be complementary to the beginning of exon 2. The goal is to amplify cDNA but not genomic DNA. If this is not possible, the RNA sample should be treated with RNase-free DNAse prior to reverse transcription.

5) BLAST your primer sequences. Run a similarity search of primers against whole organism/non-redundant databases to determine if primers might anneal to other (unwanted) targets.

6) Run positive and negative controls with conventional endpoint PCR. The positive control (using a template that has 100% similarity to the two primers) should produce a single product of the expected size after 40 cycles using a 60 oC annealing temperature and a 30 second extension time. The negative (no template) control should not produce any products after 40 cycles using a 60 oC annealing temperature and a 30 second extension time.

7) Run positive and negative controls with qPCR. It is important to establish that cDNA has been synthesized and gives a signal much earlier than DNAsed RNA that has not been subjected to reverse transcription. Small amounts of genomic DNA can lead to results that can't be used.