7500 sequence detection system

Total RNA was extracted from cells, tissues and clinical samples using Trizol reagent (Life Technologies, Carlsbad, CA, USA). To quantify the amount of target mRNA, miRNA and circRNA, cDNAs were synthesized with the PrimeScript RT Master Mix (Takara, Dalian, China). Quantitative analysis of gene expression was conducted using an Applied Biosystems (Grand Island, NY, USA) 7500 Sequence Detection System with the SYBR Premix Ex Taq Ⅱ (Takara, Dalian, China), and gene expression was calculated relative to the internal control GAPDH through the ^ΔΔCt method. The relative target gene levels were presented as the ratio of change versus internal control. The specific primers for the detected genes were listed in Table S1.

For qRT-PCR, 1 µg of total RNA used in the previous RNA-Seq library construction was used for cDNA synthesis. A PrimeScript RT enzyme with a gDNA eraser (Takara, Japan) was used for cDNA synthesis. qRT-PCR was performed on an Applied Biosystems 7500 Sequence Detection System using SYBR Premix Ex Taq™ II (Takara, Japan). The primers in this step are listed in Supplementary Table S1. The polypyrimidine tract-binding protein (CsPTB1) gene was used as an internal control^{28 (link)}. The relative expression levels were calculated using the 2^−ΔΔCt method^{29 (link)}.

Total RNA was extracted from the tissues and cells using RNeasy and miRNeasy Kits (Qiagen). One μg of total RNA was reverse-transcribed to cDNA using the Superscript II Reverse Transcriptase Kit (Invitrogen), and reverse transcription reaction were performed according to the manufacturer’s instructions. PCR was performed on an Applied Biosystems 7500 Sequence Detection System using a SYBR Premix Ex Taq GC kit (Takara). The primers were: miR-9: 5′-TCTTTGGTTATCTAGCTGTATGA-3′ (sense), 5′-TGGTGTCGTGGAGTCG-3′ (antisense); U6: 5′-CTCGCTTCGGCAGCACA -3′ (sense), 5′-AACGCTTCACGAATTTGCGT-3′ (antisense); E-cadherin: 5′-AAAGGCCCATTTCCTAAAAACCT-3′ (sense), 5′-TGCGTTCTCTATCCAGAGGCT-3′ (antisense); GAPDH: 5′-CGACCACTTTGTCAAGCTCA-3′(sense), 5′-AGGGGAGATTCAGTGTGGTG-3′ (antisense). The gene expression was normalized to the level of GAPDH using the relative ^ΔΔC_T method.