Riboex

Total RNA was isolated with RiboEX (Invitrogen) according to the manufacturer’s supplied protocol. cDNA was synthesized from the total RNA using the reverse transcriptase (Fermentase) using oligo-dT and random hexamer primers. Transcript levels were measured in duplicate by quantitative reverse transcription PCR using SYBR green (ABI step one Real-Time PCR System). Expression levels were calculated relative to B2M using the ΔΔCT method.

The total RNA was extracted using RiboEX (Invitrogen). Afterward, using a miRNA reverse transcription kit, the RNA had been reversed transcribed to cDNA. The miR-223 expression was determined utilizing a Hairpin-itTM miRNAs qPCR kit (GenePharma) and an ABI PRISM 7000 tool (Applied Biosystems, Waltham, MA, USA). As endogenous controls, U6 and GAPDH were employed. Predenaturation was carried out at 95°C for 30 s followed by 40 cycles for 5 s at 95°C for 5 s and 60°C for 30 s. 2^−ΔΔct was used to calculate the relative expression. The primers used in this research are listed as follows: GAPDH forward, 5′-CATACCAGGAAATGAGCTTG-3′, and reverse, 5′-ATGACATCAAGAAGGTGGTG-3′; U6 forward, 5′-CAAATCGGAGCATCGTGAATGCC-3′, and reverse, 5′-CAATACCATGCCGCCCAGGTC-3′; miR-223 forward, 5′-GGCAGCACCCCATAAACTGTT-3′, and reverse 5′-CAGTGCGTGTCGTGTCGTGGAG-3′; and RhoB forward, 5′-CGAATCCATTCAAATCCGGGATCCG-3′, and reverse 5′-AAGGCATTCAATCGGACTTACv3′.