2.9 Real-time PCR
RS-NC-mediated gene silencing was measured using real-time PCR of cDNA.
First, 1.0 × 105 MDA-MB-231 cells were seeded to wells
of a 6-well plate and incubated for 16 hours at standard conditions.
Then, 200 nM Kaiso siRNA was incubated with RS peptide of N/P ratio 5 in
2 mL of total volume with Opti-MEM at room temperature for 30 minutes.
After four-hour incubation with RS peptide, the buffer was changed by
DMEM and incubated for 20 hours at standard conditions. Next, cells were
rinsed with NFW three times, and the total RNA of the cells was purified
by following the manufacturer’s protocol of Tri-RNA solution (Biotech
Co., Kaohsiung, Taiwan). Absorbances at 260 and 280 nm of purified RNA
were measured using the Take3 micro-volume plate and Epoch2 microplate
spectrophotometer. Then, according to the manufacturer’s protocol, cDNA
of total RNA 100 ng was synthesized using the ReverTra Ace® qPCR RT
Master Mix and gDNA Remover kit (Promega, WI, USA). The qPCR reaction of
synthesized cDNA was performed by following the manufacturer’s protocol
of GoTaq® qPCR Master Mix (Promega, WI, USA) in the QuantStudio 3
real-time PCR system (Applied Biosystems, CA, USA). The thermal cycle
was composed of pre-denaturation at 95°C for 60 seconds, denaturation at
95°C for 15 seconds, annealing at 55°C (β-actin), 53.2°C (KAISO), 52.4°C
(LIPIN1), 51.2°C (PICH), 58.9°C (PML) for 30 seconds, and extension at
72°C for 60 seconds. The relative expression of each mRNA was calculated
using the ΔΔCt method and normalized by the mRNA expression level of
β-actin.