Taqman reverse transcription reaction

Specific quantitative assays for mRNAs for miR-21 targets were carried out using SYBR Green dye. The sequences of all the primers pairs used are given in Table 1. RNA samples were treated with DNase to eliminate potential genomic DNA contamination. Complementary DNA (cDNA) was synthesized by using 1 µg total RNA from each sample and random hexamers in a Taqman reverse transcription reaction (Applied Biosystems, Foster City, CA, USA). 10 ng cDNA and gene-specific primers were added to SYBR Green PCR Master Mix (SYBR Green I Dye, AmpliTaq-DNA polymerase, dNTPs mixture dUTP and optimal buffer components (Applied Biosystems, Foster City, CA), and subjected to PCR amplification (one cycle at 50 °C for 2 min, one cycle at 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min). Real time PCR was performed on an ABI 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). The data were collected and analyzed with Sequence Detection Software 2.0 (Applied Biosystems, Foster City, CA). The resulting amplicon products were visualized on an agarose gel to verify size and specificity of RT-PCR reaction. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. Relative gene expression was determined using the delta-delta CT method (Pfaffl et al., 2002 (link)).