Sybr green rox qpcr mastermix

The epithelium of each leukoplakia specimen was dissected using laser capture microdissection. Total RNA extracted from each tissue with RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) was reverse transcribed using iScript complementary DNA (cDNA) synthesis kit (Bio-Rad, CA, USA). Real-time polymerase chain reaction (RT-PCR) was performed using the SYBR Green/ROX qPCR Mastermix (SABiosciences, Frederick, MD, USA) on the ABI Prism 7000 Sequence Detection System (Perkin Elmer Applied Systems, Foster City, CA, USA). Each reaction contains 2 × 12.5/μl of SYBR Green Mastermix, 1 μl of 10 μM of primers and 50 ng of the cDNA, to a total volume of 25 μl. The thermal cycling conditions included an initial denaturation step at 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s, annealing temperature for 30 s and extension at 72°C for 30 s. Amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and small proline-rich protein 2a (SPRR2a), a gene abundantly expressed in epithelial cells was included as controls for the PCR reaction and epithelial cell origin respectively. The primers used are given in Table 1. The magnitude of change in the messenger RNA was expressed as 2⁻ΔΔCt.[29 (link)]

Paired oligonucleotide forward and reverse primers (Table S2) for C-X-C chemokine receptor type 4 (CXCR4) and GAPDH were designed using Primer Designer (Scientific and Educational Software, Durham, USA) against the sequence downloaded from GenBank and obtained from Invitrogen. The process of RNA extraction and cDNA generation was the same as those in the PCR array experiment. The cDNA sample was mixed with forward and reverse primers and SYBR Green ROX qPCR Mastermix (QIAGEN, West Sussex, UK). The PCR mixture was processed and analyzed with the Rotor-Gene Q system (QIAGEN, West Sussex, UK). All mRNA data were expressed relative to the endogenous control gene, GAPDH.