Cq values and the logarithm of RNA concentrations
(ng) were linearly correlated for each ofthe examined genes in the range
1000–0.1 ng.
A similar reaction efficiency, included in the optimal range 90–110%,
was assessed for the housekeeping gene (-tubulin) and the four target genes.
As cDNA synthesized from 100 ng of total RNA was efficiently amplified, this concentration
was utilized in the subsequent real-time PCR reactions.
The melting curve
analysis showed the presence of a single melting peak for each of the tested genes,
indicating that all primer pairs reported in Table 1 amplified a single product with a
distinct melting temperature (data not shown).
In all negative-control samples, no
fluorescent signal was detected, proving that there were no contaminations in the
reaction mixtures and that the DNA traces were effectively removed.