Designing primers for quantitative real time (q-PCR)
An excellent and fast way to select primers is with the free onlinetool Primer3, currently in v0.3. Primer3Plus, a variation of Primer3 has qPCR
settings.
Or just apply the following or similar settings to Primer3:

pair towards 3' end (often more specific, some cDNAs don't contain)

pair separated by an exon-exon boundary (reduces genomic background)
e.g. last exon & penultimate

amplified region must be no bigger than 200 bp; usally 60-150 bp

GC content: 50-60%

min length: 18, max length 24 (best: 20 nt)

melting temperature: min 60, max 63, best 60

max Tm difference: 10 (shouldn't be more than 1 in final pair) ??

max 3' self complementary: 1

max poly-x: 3
Verify by blasting the primers sequences. Target gene should come out
with the lowest E value. No other gene should be close. Also check whether
possible isoforms will be detected by the candidate primer pair.
qPCR primers vs probes
When designing primers:
· Tm should be within 58°C to 60°C
· the last 5 bases at the 3 prime end should have no more than two G's or C's
· keep the annealing Ts of the primers as close as possible
· select primer pairs with minimal number of potential primer dimers and
primer hairpins as possible
When designing probes:
· Tm should be within 68°C to 70°C
· no Gs on the 5’ end
· select the strand that gives the probe more C than G bases
· make the TaqMan MGB probes as short as possible, without being shorter
than 13 nucleotides.
Before to proceeding with real time PCR, it is necessary to test the primers
on a PCR reaction to ensure that the primers amplify the gene of interest at
the right size and that the primers are specific (i.e. no other bands present on
the gel except that of the expected size).
In doing this, it is important to run your test PCR under conditions as close as
possible to those for real time PCR (refer to the table below).
The best results are achieved with magnesium concentration at 3mM
and with 0.025 U/ul of Taq.
Table 4: Typical Real Time PCR stage Parameters