Ultrapure rna kit

Total RNA was extracted using the Ultrapure RNA Kit (CWBio, Beijing, China), and the extracted RNA was then used as the template for a reverse transcription with the First Strand cDNA Synthesis Kit (CWBio). The relative RGS4 expression level was determined in a qRT-PCR assay using the UltraSYBR Mixture (CWBio). Relative mRNA levels were calculated according to the 2^−ΔΔCt method and were normalized to GAPDH levels. The following primers were used for qRT-PCR: RGS4-F (5′-GCC AAG AGG AAG TCA AGA AA-3′), RGS4-R (5′-CTT CAC AGC TGA TCC AGA AG-3′), and GAPDH-F (5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′) and GAPDH-R (5′-AGC CTT CTC CAT GGT GA A GAC-3′).

Total RNA extracted from transfected MCF-7 and MDA-MB-231 cells with an Ultrapure RNA kit (CWBio) were reverse transcribed into cDNA at 42°C for 50 min and 85°C for 5 min with the miRNA cDNA Synthesis kit (CWBio), following the manufacturer's manual. RT-qPCR analysis was then performed following the instructions of the miRNA qPCR Assay kit (CWBio). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min; and final extension at 72°C for 50 sec. miR-653-5p and MAPK6 expression levels were calculated using the 2^−∆∆Cq method (16 (link)) and normalized to the internal reference genes U6 and GAPDH, respectively. The following primers were used for qPCR: miR-653-5p forward, 5′-GTGTTGAAACAATCTCTACTG-3′ and reverse, 5′-GAACATGTCTGCGTATCTC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′; MAPK6 forward, 5′-AGCGCTAGAGGAAGCATCAC-3′ and reverse, 5′-GTGGGATGCCTATGGACTCG-3′; and GAPDH forward, 5′-TATGATGATATCAAGAGGGTAGT-3′ and reverse, 5′-TGTATCCAAACTCATTGTCATAC-3′.