Ab7300 thermo recycler

Total RNA was isolated from the tumor tissues or cells using TRIzol Reagent (Invitrogen) following the manufacturer’s protocol. The purity and concentration of the RNA samples were examined by measuring the absorbance at 260, 280, and 230 nm using the NanoDrop ND-100 (Thermo Fisher Scientific). Specifically, optical density (OD)260/OD280 ratios between 1.8 and 2.1 were deemed acceptable, and OD260/OD230 ratios greater than 1.8 were deemed acceptable.
RNA was reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). Then qRT-PCR was performed using an AB7300 thermo-recycler (Applied Biosystems) with primers (Gene Pharma) and the TaqMan Universal PCR Master Mix. GAPDH was used as the reference gene for FGF18 mRNA.
The primers used to value FGF18 expression were:
The GAPDH primers were:

Trizol solution (Invitrogen) was employed to extract RNA from tissues and cells, and complementary DNA (cDNA) was synthesized by Prime Script RT Master Mix (Thermo Fisher Scientific). QRT-PCR for circ-ITCH, miR-106a, or CDH1 was performed on AB7300 thermo-recycler (Applied Biosystems, Foster City, CA, USA) using SYBR Select Master Mix (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal parameter of circ-ITCH and CDH1, and U6 acted as an internal control for miR-106a. The sequences were listed as follows: circ-ITCH, forward: 5′-AGGATCCCAGGAGTTCAAAT-3′; reverse: 5′-GAGTGGGCTTGACTGAAATAG-3′. GAPDH, forward: 5′-TATGATGATATCAAGAGGGTAGT-3′; reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′. CDH1, forward: 5′-ACACCATCCTCAGCCAAGA-3′; reverse: 5′-CGTAGGGAAACTCTCTCGGT-3′. MiR-106a, forward: 5′-GAGAACAGCAGGTCCAGCAT-3′; reverse: 5′-CTTCCT CAGCACAGACCGAG-3′. U6, forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′-AACGCTTCACGAATTTGCGT-3′. The relative level was analyzed by the 2^−∆∆Ct method.

Total RNA was isolated from tissues and cells by using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. cDNA was synthesized using HiScript II (Vazyme, China) and qRT-PCR for circRNA and miRNA was performed on AB7300 thermo-recycler (Applied Biosystems, USA) or LightCycler 480 (Roche, USA) using primers (Sango Biotech, China) listed in Additional file 1 Table S1. U6 and β-actin were used as an internal standard control for miRNA and mRNA detection, respectively. Each sample was replicated three times and data was analyzed by comparing Ct values.

Total RNA was isolated from tissues by using TRIzol reagent (Invitrogen, USA). Then, cDNA was synthesized with HiScript II (Vazyme, China) for qRT-PCR. Primers for qRT-PCR, which was conducted on an AB7300 thermo-recycler (Applied Biosystems, USA) or LightCycler 480 (Roche, USA), were provided by TSINGKE Biological Technology. U6 and beta-actin were used as the internal reference. Expression levels of circRNAs were calculated using the 2^−ΔΔCT method.

Total RNA was isolated from cells using TRIzol reagent (Invitrogen, USA). Total RNA was reversed to cDNA using HiScript III Reverse Transriptase (Vazyme, Nanjing, China). QRT-PCR was performed on an AB7300 thermo-recycler (Applied Biosystems, USA) using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) to amplify cDNA with specific primers (Table 1), GAPDH and β-actin were used as an internal standard control, respectively.Table 1

Sequences of the primers utilized for real-time quantitative PCR

Name	Sequence
CRYAB
Forward (5′–3′)	CCTGAGTCCCTTCTACCTTCG
Reverse (5′–3′)	CACATCTCCCAACACCTTAACTT
TFRC
Forward (5′–3′)	AGGTGTTGGGAGATGTGATTGA
Reverse (5′–3′)	GGATGAAGTAATGGTGAGAGGGT
SQLE
Forward (5′–3′)	GGCATTGCCACTTTCACCTAT
Reverse (5′–3′)	GGCCTGAGAGAATATCCGAGAAG
G6PD
Forward (5′–3′)	CGAGGCCGTCACCAAGAAC
Reverse (5′–3′)	GTAGTGGTCGATGCGGTAGA
GAPDH
Forward (5′–3′)	TGTGGGCATCAATGGATTTGG
Reverse (5′–3′)	ACACCATGTATTCCGGGTCAAT
β-actin
Forward (5′–3′)	CATGTACGTTGCTATCCAGGC
Reverse (5′–3′)	CTCCTTAATGTCACGCACGAT

We extracted the total RNA by using TRIzol reagent (Invitrogen) and decided the concentration via the NanoDrop ND-1000. The primers for the detection of circHIPK3, miR-107, and BDNF were designed and purchased from GenePharma. qRT-PCR was applied using an AB7300 thermo-recycler (Applied Biosystems, Carlsbad, CA) with a TaqMan Universal PCR Master Mix. The relative expression of targets was determined by the 2^−ΔΔCt method.