Abi 7900 pcr system

Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed to first-strand cDNA using a TaqMan Reverse Transcription Kit, according to the manufacturer’s protocols (Applied Biosystems, Carlsbad, CA, USA). Primers were synthesized by Sangon (Shanghai, China) (Table S2), and PCR assays were performed in triplicate using Real-Time PCRBIO SyGreen Mix and an ABI7900 PCR system (Applied Biosystems) and averaged. For each sample, differences in the threshold cycle (ΔCt) values were calculated by correcting the Ct of the target genes to the Ct of the reference gene β-actin (ACTB), and the relative gene expression was expressed as 2^−ΔΔCt with respect to the control group.⁷⁴ (link) For miR-140 quantification, MicroRNA Purification Kit (Norgen, Thorold, ON, CA) and Bulge-loop miRNA qRT-PCR Primer Sets (one reverse transcriptase primer and a pair of qPCR primers for each set) specific for miR-140 (RiboBio) were used, according to the manufacturer’s protocols, and small nuclear RNA (snRNA) U6 was utilized as an internal control.

Total RNA was isolated from tissues using RNeasy (Qiagen, Hilden, Germany), and RNA quality was analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number of the samples was ≥7.6. cDNA was synthesized with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). The gene expression profile was analyzed with the Human Extracellular Matrix and Adhesion Molecules RT 2 Profiler PCR Array (PAHS-013, SABiosciences, Frederick, MD, USA) on an ABI 7900 PCR system (Applied Biosystems).

Total RNAs were isolated form tissues, cells, exosomes using Trizol (Invitrogen), and A260/A280 was determined by an ultraviolet spectrophotometer. RNA concentration (μg/μL)  =  (A260 × 40 ×  dilution factor)/1000. The purity should be 1.8–2.1. For WDR82, cDNA was collected from RNA (2 μg) through first-strand cDNA synthesis kit (Thermo Fisher Scientific) while for miR-155-3p, that was collected through NCode miRNA first-strand cDNA kit (Invitrogen). Real-time PCR was performed on the ABI7900 PCR system (Applied Biosystems, CA, USA) using SYBR Green PCR Master Mix (Takara, Dalian, China). The loading control of miR-155-3p was U6, and that of WDR82 was β-actin. The relative expression was calculated by 2^−△△Ct method and the primer sequences are shown in Additional file 1: Table S1.

RNA isolation from cells and tissues was performed using the TRIzol reagent (Takara, Dalian, China). The reverse transcription of miRNAs into cDNA was performed using the MiR-X miRNA First Strand Synthesis kit (Clontech, USA). For the mRNA detection, the first strand cDNA synthesis kit (Takara) was used to transcribe mRNA into cDNA. The real-time PCR was performed on an ABI7900 PCR system (Applied Biosystems, Foster City, USA) using TB Green Premix EX Taq II kit (Takara). The U6 and β-actin were used as internal controls to normalize miR-140-5p and survivin expression levels, respectively. The 2^−ΔΔCt method was used to calculate the differential expression of miRNA and mRNA.