Premix ex taq 2 kit

Total RNA was extracted from the clinical samples and cell lines with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions. The cDNA was synthesized by One Step PrimeScript cDNA Synthesis Kit (Takara, Shiga, Japan). QRT-PCR was performed by Premix Ex Taq II kit (Takara), on an ABI 7600 fast Real-time PCR System (Applied Biosystems). The All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia, Rockville, MA, USA) was used for the determination of miR-346. The relative expression fold changes were calculated using the 2^−ΔΔC_t method. GAPDH or U6 were tested as the internal controls. CircSMARCA5 5′-ACA ATG GAT ACA GAG TCA AGT GTT-3′, 5′-CCA CAA GCC TCC CTT TTG TTT T-3′; GAPDH 5′-TGT TCG TCA TGG GTG TGA AC-3′, 5′-ATG GCA TGG ACT GTG GTC AT-3′; miR-346 5′-TGT CTG CCC GCA TGC CTG CCT CT-3′; U6 5′-CTC GCT TCG GCA GCA CA-3′, 5′-AACGCTTCACGAATTTGCGT-3′. The experiment was performed in triplicate.

miR-29a-3p Oligod (T) specific RT primers were designed. Cells were dissolved in 1 mL Trizol (Thermo Fisher Scientific, Waltham, MA, USA) to extract the total RNA based on instructions. RNA samples in which the miR-29a-3p expression was detected were treated with 3’ poly-A tail. RNAs were reversed transcript into cDNA using M-MLV reverse transcriptase (D7160L, Beyotime, Shanghai, China) and random primers. The PCR reaction system and reaction conditions were prepared based on the instructions of Premix Ex Taq™II kit (Takara, Dalian, China). ABI7500 quantitative PCR apparatus (Applied Biosystems, Shanghai, China) was used for RT-PCR with U6 as internal control of miRNA and GAPDH of mRNA. Data were analyzed using 2^-ΔΔCt method ^{22 (link)}: ΔΔCt = [Ct_{(target gene)}-Ct_{(internal gene)}]_{experimental group} -[Ct_{(target gene)}-Ct_{(internal gene)}]_{control group.} The primers for gene are listed in Table 1.

Total RNA from CC tissues and cell lines were isolated by TRIzol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. NanoDrop-2000 spectrophotometer was used to confirm the quantity and quality of the extracted RNA. RNA was reversely transcribed by M-MLV (Promega, Madison, WI, USA), and then Premix ExTaq II Kit (Takara) was used to perform qPCR. The relative fold changes of candidate genes in the transcripts were analyzed with 2^−ΔΔCt method. The specific primers are shown in Table 1.

Total RNA form cells (HaCaT, SCC13 and Colo16) and tissues (human tumor, human normal, and mice xenograft model tumor) was extracted with Trizol reagent (Invitrogen). Next, complementary DNA (cDNA) was synthesized by One Step PrimeScript cDNA Synthesis Kit (Takara, Shiga, Japan) and microRNA First-Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China). Then, qRT-PCR reactions were performed using the Premix Ex Taq II kit (Takara) on 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). In this study, primers used for amplification were listed as follows: circSEC24A (Forward, 5ʹ-GCTCTCCTTAAACAGGATATACACAA-3ʹ; Reverse, 5ʹ-TGTCCACTGAGAAGGAATAAGTCA-3ʹ); SEC24A (Forward, 5ʹ-TTCCTTCTCAGTGGACAGTATTC-3ʹ; Reverse, 5ʹ-GCTGATGATGGTAAGAGGGATAG-3ʹ); miR-1193 (Forward, 5ʹ-ACACTCCAGCTGGGATAGACCGGTGACGTGC-3ʹ; Reverse, 5ʹ-CTCAACTGGTGTCGTGGA-3ʹ); MAP3K9 (Forward, 5ʹ-ATGCAAGATTTGGCTGTCCTTC-3ʹ; Reverse, 5ʹ-TGGTAGTAATATGAGAACTAC-3ʹ); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Forward, 5ʹ-GGAGAAGCTGAGTCATGGGT-3ʹ; Reverse, 5ʹ-GGTGGGAGATCTGGTTTCCG-3ʹ), U6 (Forward, 5ʹ-GTGCGTGTCGTGGAGTCG-3ʹ; Reverse, 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ). The relative expression of circSEC24A, SEC24A, MAP3K9, and miR-1193 was evaluated with 2-^ΔΔCt method, and the expression of circSEC24A, SEC24A and MAP3K9 was normalized by GAPDH and miR-1193 level was normalized by U6.