Lightcycler faststart dna master sybr green system

BMDMs (10⁵ cells/assay) were infected with T. whipplei as previously described [15] (link). BMDMs were lysed, and DNA was extracted using a QIAamp DNA MiniKit (Qiagen). PCR was performed using a LightCycler-FastStart DNA Master SYBR Green system (Roche) and was conducted with primers specific for the T. whipplei 16S-23S ribosomal intergenic spacer region (tws3f and tws4r), as described previously [33] (link). In each PCR run, a standard curve was generated using serial dilutions ranging from 10 to 10⁸ copies of the intergenic spacer region and established by the LightCycler 5.32 software (LC-Run version 5.32; Roche) as previously described [15] (link).

Complementary DNA (cDNA) synthesized from the extracted total RNA was used to detect the expression of the Bax and Bcl-2 genes using specific oligonucleotide primers (Table 1).
The reaction mixture comprised 10 µl, containing 5 µl of prime Q-Master Mix with SYBR Green I (Pars Tous, Iran), 1.1 µl of cDNA, 1 µl of forward and reverse primers and 2.9 µl of sterile deionized water, according to the manufacturer's instructions.
Real-time-PCR was performed using the LightCycler-FastStart DNA Master SYBR Green system (Roche Molecular Biochemicals, Mannheim, Germany) with a 15-minute preincubation at 95 °C, followed by 55 cycles of 60 seconds at 58 °C (denaturation) and 10 seconds at 95 °C (annealing). The products were analyzed for a melting curve using the light cycler system to avoid nonspecific product amplification. The β-actin gene with the same annealing temperature was used as an internal control for housekeeping gene expression. All reactions were performed in duplicate.