Sybr premix ex taq 2

Primers for the coding genes and internal controls (Additional file 1) were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Total RNA was converted to cDNA by using a PrimeScript™ RT Reagent Kit containing gDNA Eraser (TAKARA, Dalian, China), and oligo (dT) and random hexamer primers. The q-PCR was performed using SYBR Premix Ex Taq™ II (TAKARA). The reaction mix composed of 1μL template cDNA, 0.4μL of 10μM forward and reverse primers, 5μL SYBR Premix Ex Taq™ II, and 3.2 μL dH2O at a final volume of 10 μL. The reactions were performed on a Rotor gene 6000 PCR System (QIAGEN, Hiden, Germany) as follows: 95°C for 10 s, followed by 40 cycles of 95°C for 5 s, and 20 s at the Tm (Additional file 1). Melting curve analysis was performed from 65°C to 95°C in 1.5°C increments. The expression levels of genes were normalized to HPRT1 and GAPDH. Relative gene expression levels were calculated using the 2^−ΔΔCt method33 (link) and data were expressed as mean ± standard error of the mean (SEM).

Nuclear and cytoplasmic separation was performed as per the protocol of the PARIS kit (Invitrogen) to determine circRNA localization. Total RNAs was prepared using the RNeasy mini kit according to the standard protocol. To validate the cyclization of circRNA, 2 μg of RNA extractions was incubated with RNase R (3 U/μg, Epicenter Technologies) for 1 h at 37°C. Thereafter, the first‐strand of cDNA was synthesized using the PrimeScript RT polymerase (Qiagen) or miScript II RT kit (Qiagen), and quantitative PCR was then implemented using the SYBR Premix Ex Taq II (Qiagen). Fold changes were assessed using relative quantification (2^−ΔΔCt) with U6 or glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as a reference control. The primer sequences were:

circ_0010235: F 5′‐GGGCCTGTACTGTCTGTGTA‐3′, R 5′‐CTCCCACCCTCCCATTACCTT‐3′;

E2F7: F 5′‐GCAGTGGTTGTTTCTGTCAGG‐3′, R 5′‐AACCCTGGTCAGTGTAGGGC‐3′;

ALDH4A1: F 5′‐CTCAGCCTTCGAGTACGGTG‐3′, R 5′‐CCCGAAGATCTCCTTGGCAT‐3′.

GAPDH: F 5′‐TCACCACCATGGAGAAGGC‐3′, R 5′‐GCTAAGCAGTTGGTGGTGCA‐3′;

miR‐379‐5p: F 5′‐GCCGAGTGGTAGACTATGGAA‐3′, R 5′‐CTCAACTGGTGTCGTGGA‐3′;

U6: F 5′‐CTCGCTTCGGCAGCACA‐3′, R 5′‐AACGCTTCACGAATTTGCGT‐3′.