Melting curve data were obtained using FRET as described in detail by You and coworkers [49 (link)]. Briefly, DNA or RNA oligonucleotides labeled with Cy3 or TYE563 at the 3’-end (hereafter designated as “target”) were purchased from DNA Technology (Denmark) or Sigma-Aldrich (USA). Complementary oligonucleotides (hereafter designated as “probes”) were labeled in-house with FITC at the 5’-end or purchased with a 5’ FAM label from Exiqon A/S (Denmark). Hybridization was quantified by FRET between the FITC and Cy3 labels or the FAM and TYE563 labels (detected as a decrease in FITC or FAM fluorescence) when they were brought in close proximity due to the formation of a duplex between the probe and its target. The target oligonucleotides were used at concentrations of 25, 50, 100, 200 and 400 nM, and the probe was always used at a concentration of 50 nM. The probe-target interactions were measured in a 384-well optical plate in a volume of 20 μl in buffer containing 150 mM NaCl and 50 mM Tris-HCl (pH 8.0) using an ABI7900HT Real-Time PCR instrument (Life Technologies, USA). The temperature was rapidly increased to 95°C, and the complexes were allowed to melt for 10 min. Then, the temperature was decreased to 5°C over 45 min, stabilized for 5 min and increased slowly (over 45 min) to 95°C. The Tm values were calculated from the obtained melting curves.
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