Fast sybr green pcr master mix kit

The total RNA from three human OSCC cell lines was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and these RNA extracts (2 μg) were further reverse transcribed into first-strand complementary DNA (cDNA) using SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) by following the manufacturer’s instructions. The mRNA expression levels of MMP2 were next analyzed using a pair of oligo primers which were described as follows: MMP2: Forward, 5′- TGATCTTGACCAGAATACCATCGA-3′; Reverse, 5′- GGCTTGCGAGGGAAGAAGTT-3′. GADPH: Forward, 5′-TGCACCACCAACTGCTTAGC-3′; Reverse, 5′-GGCATGGACTGTGGTCATGAG-3′. One twentieth of the complementary DNA generated was used as a template, and gene amplification and detection were achieved using a Fast SYBR^® Green PCR Master Mix kit (Applied Biosystems; Life Technologies, Inc., Vilnius, Lithuania) in a StepOnePlus™ Real-Time PCR System (Applied Biosystems; Life Technologies, Inc.).

Quantitative RT-PCR (RT-PCR) was performed by real-time analysis using the Step One Plus detection system (Applied Biosystems) and FAST SYBR GREEN PCR Master Mix kit (Applied Biosystems). RNA was isolated and purified using the Trizol extraction method (Invitrogen). cDNA was then generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).