Dtlite 4 real time pcr system

Three complementary duplexes were prepared to compare the effects from single-stranded oligonucleotides and their double-stranded forms. They included duplexes of tRNA with _CtRNA, rrs₋₁₁₅ with _Crrs₋₁₁₅ and ProA-ter with rrl_Pr. Oligonucleotides from stock solutions were mixed in equimolar concentrations (20 μl), melted and annealed in DTlite 4 Real-Time PCR System (DNA Technology, Russia) according to the following program: heating from RT at a rate of 1°C/0.8 min; melting for 5 min at 75.2°C in the case of tRNA/_CtRNA duplex and for 5 min at 56°C in the case of rrs₋₁₁₅/_Crrs₋₁₁₅ and ProA-ter/rrl_Pr duplexes; cooling to storage temperature (4°C) at a rate of 1°C/10 min. Single stranded oligonucleotides and their duplex were electrophoretically analyzed in 15% PAGE at 150V using 50+ bp DNA Ladder (Evrogen, Russia) as markers. Freshly prepared duplexes were added to the bacterial cultures to a final duplex concentration of 2 nmol.

Real-time PCR was done for the following SNPs: rs 9594782 (C > T), 2277438 (G > A) for RANKL polymorphisms, rs 1805034 (C > T), 1245811 (A > G), and 75404003 (C > DEL) for RANK polymorphisms, and rs 207318 (G > C) for OPG polymorphism. It was carried out on DT lite 4 Real-Time PCR System (DNA Technology, Russian) using the following program: one cycle of incubation at 50 °C for 2 min then one cycle for 10 min at 95 °C and lastly 40 cycles of incubation at 95 °C fo 00:15 and then for 1:00 min Results were interpreted on software DT lite 4 7.9