Mirna reverse transcription kit

Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Reverse transcription was performed using the miRNA Reverse Transcription Kit (Qiagen) or GoScript Reverse Transcription System (Promega, Madison, WI, USA). The obtained complementary DNA was used with the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) for qRT-PCR in the ABI7500 system (Applied Biosystems). All primer sequences were listed in Table 2. Relative expression was figured by the 2^−ΔΔCt method and normalized to β-actin or U6 small nuclear RNA (U6).Table 2

Primer Sequences for RT-qPCR

Genes	Primer Sequences (5ʹ-3ʹ)
circDNM3OS	Forward (F): 5ʹ-CTGCTGAGAAAAGACTGCCGA-3ʹ
	Reverse (R): 5ʹ-ACCTAGGTTCCCTTGGTCACA-3ʹ
MORC2	F: 5ʹ-GGAGGTTCCTTCTCCCAAAGTC-3ʹ
	R: 5ʹ-CAGAAACTGCGACACTCCGCTT-3ʹ
miR-145-5p	F: 5ʹ-GTCCAGTTTTCCCAGGA-3ʹ
	R: 5ʹ-GAACATGTCTGCGTATCTC-3ʹ
DNM3OS	F: 5ʹ-GGTCCTAAATTCATTGCCAGTTC-3ʹ
	R: 5ʹ-ACTCAAGGGCTGTGATTTCC-3ʹ
β-actin	F: 5ʹ-AAATCTGGCACCACACCTTC-3ʹ
	R: 5ʹ-GGGGTGTTGAAGGTCTCAAA-3ʹ
U6	F: 5ʹ-GCTCGCTTCGGCAGCACA-3ʹ
	R: 5ʹ-GAGGTATTCGCACCAGAGGA-3ʹ

Quantitative real‐time polymerase chain reaction (qRT‐PCR) assay was employed to evaluate RNA expression. Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used isolate RNA from melanoma tissues and cell lines. To detect the expression level of circRNA, mRNA and miRNA, total RNA was reverse transcribed using iScript cDNA Synthesis Kit (Bio‐Rad, Shanghai, China) or miRNA Reverse Transcription Kit (QiaGen, Valencia, CA, USA). QRT‐PCR was carried out by using SYBR green PremixEx Taq II (Takara, Dalian, China). In this study, the 2^−ΔΔCt method was carried to evaluate RNA levels. The primer sequences were provided in Table 1.

The total RNA from tissues and cells was extracted directly using the miRNeasy Mini Kit (QIAGEN, USA). Briefly, tissues or cells were collected in a reaction tube, lysed with 700 µl QIAzol and mixed with 140 µl chloroform. After being centrifuged at 12,000g for 15 min at 4 °C, the upper aqueous phase was transferred to an RNeasy Mini spin column in a 2-ml collection tube and mixed with 100% ethanol. After being washed with 700 µl Buffer RWT and 500 µl Buffer RPE, the total RNA was collected and quantify by NanoDrop 3000 (Life Technologies). For extraction and quantification of miR-338-3p and miR-3065-5p in the collected serum, 1 mL serum was used, and cel-miR-39-3p (hereafter as spike-in, 20 fmol spike-in/200ul serum) was used as spike-in control. Serum were mixed with 500uL of QIAzol and followed by the protocol described above. All miRNA samples were reverse-transcribed and quantified by miRNA Reverse Transcription Kit (QIAGEN) and miScript SYBR Green PCR Kit (QIAGEN). For quantification of miRNA in cells or tissues, U6 was used as control, and for miRNA in serum, spike-in was used as internal control.

MicroRNA was extracted using the miRNeasy Mini Kit (QIAGEN) and reverse transcription was performed with a miRNA Reverse Transcription Kit (QIAGEN). The relative expression levels of miR-21 in tissues from patients and cell lines were detected using the miScript SYBR Green PCR Kit (QIAGEN), with U6 as the internal control (12 (link)). The primers used in the present study were listed in Table SI. The PCR program consisted of three steps: denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec, with 40 cycles in total. For the detection of other genes, total RNA was isolated with TRIzol^® (Invitrogen) and reverse transcription was performed with the PrimeScrip RT reagent Kit with gDNA Eraser (TaKaRa Bio). Real-time qPCR was performed using PrimeScript RT Master Mix, with GAPDH as the internal control. The cycling program consisted of one cycle at 95°C for 1 min, and 40 cycles of 15 sec at 95°C, 20 sec at 55°C, plus 60 sec at 72°C. Each sample was evaluated in triplicate. Default threshold settings were used as threshold cycle (Ct) data. Relative quantification of gene expression was performed using 2^−ΔΔCt method, which indicates relative fold changes.