Tb green premix ex 2

Total RNA was extracted from lungs and cells using RNAiso Plus (TaKaRa, Tokyo, Japan). The isolated total RNA was reverse transcribed using Mir-X miRNA First-Stand Synthesis Kit (TaKaRa, Tokyo, Japan) for microRNA let-7d and the PrimerScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Tokyo, Japan) for mRNA, according to manufacturer instructions. Relative expression was assessed by ABI7500 Fast Real-Time PCR System (Applied Biosystems, USA) with the TB Green Premix Ex II (TaKaRa, Tokyo, Japan). Relative expression was calculated using the 2^−ΔΔC_T method and was normalized to the expression of U6 or GAPDH. ALL qRT-PCR reactions were performed in triplicate. The sequences of primer pairs are described in Table 1.

The cDNA was obtained by using random primers and a reverse transcription kit (catalog number RR037A, Takara). CircRNA sequences were amplified by divergent primers which were designed by Primer3 (https://primer3.ut.ee/) to cover the back-splicing sites. PCR products were extracted by agarose gel DNA extraction kit (catalog number 9762, Takara). Then the purified DNA was sent to perform Sanger sequencing. Then qPCR was performed to examine the expression levels of circRNAs on a LightCycler 96 system (Roche, Germany) using TB Green Premix Ex II (catalog number RR820A, Takara) according to the instructions. The relative expression levels were normalized to 18S.

Random primers and Reverse Transcription Kit (#RR037A, Takara) were used to obtain cDNA according to the manufacturer’s protocol. CircPrimer 2.0 software was used to annotate and obtain circRNA sequences (Zhong et al., 2018 ). Then, the divergent primers, which coved the back-splicing regions, were designed by Primer3¹ (Untergasser et al., 2012 (link)). The PCR products of divergent primers were sequenced to validate the corresponding back-splicing sites. The relative expression levels of selected circRNAs were detected by qRT-PCR using TB Green Premix Ex II (#RR820A, Takara) on a LightCycler 96 system (Roche, Germany) according to the instructions. 18S was used to normalize the threshold cycle (Ct) values, and gene expression was quantified using the relative quantitation method (2^–ΔΔCt). All experimental data are presented as means ± SD.