Mirna first strand cdna synthesis tailing reaction kit

For mRNA analysis, total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed with a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions. A Light Cycler 480 instrument (using the 384-well module) with 2×Polarsignal qPCR mix (MIKX) was used for quantitative real-time PCR analysis. Target mRNA expression levels were normalized to the expression level of β-actin in each individual sample. The 2^−ΔΔCt method was used to calculate relative expression changes. For microRNA analysis, 1 μg of total RNA was reverse transcribed with a miRNA First Strand cDNA Synthesis kit (Tailing Reaction) (Sangon Biotech) according to the manufacturer’s instructions. A Light Cycler 480 instrument (using the 384-well module) with 2×Polarsignal qPCR mix (MIKX) was used for quantitative real-time PCR analysis. U6 was used as the control for miRNA expression, and the relative expression of microRNAs was calculated by the 2^-ΔΔCt method. S1 Table lists the primers detail.

Total RNA was extracted using Trizol (15,596,026, Invitrogen). Next, total RNA was reversely transcribed into cDNA according to the instructions of PrimeScript RT reagent Kit (RR047A, Takara Bio Inc., Otsu, Shiga, Japan). The RNA was reversely-transcribed into cDNA for miRNA detection using miRNA First Strand cDNA Synthesis (Tailing Reaction) kit (B532451-0020, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China). The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted for synthetized cDNA using Fast SYBR Green PCR kit (Applied Biosystems, Carlsbad, CA) and ABI7500 qPCR instrument (ABI Company, Oyster Bay, NY). GAPDH and U6 were regarded as the internal reference to quantify relative expression using the 2^−ΔΔCt method (Additional file 1: Table S1).

The total RNA (2 μg) was extracted from the obtained tissues and cells by Trizol reagent (16096020, Thermo Fisher Scientific, Waltham, MA, USA). For miRNA detection, the total RNA was reverse transcribed into complementary DNA (cDNA) according to the instructions of miRNA First Strand cDNA Synthesis (Tailing Reaction) kit (B532453‐0020, Shanghai Sangon Biotech, Shanghai, China). For mRNA detection, the total RNA was reverse transcribed into cDNA according to the instructions of Reverse transcription Kit (RR047A, Takara Bio Inc, Otsu, Shiga, Japan). The cDNA was synthesized according to the instructions of cDNA Kit (K1622, Fermentas Inc, Ontario, CA, USA). Next, reverse transcription quantitative polymerase chain reaction (RT‐qPCR) was performed using TaqMan Gene Expression Assays protocol (Applied Biosystems, Foster City, CA, USA) with cDNA as template. β‐actin was used as an endogenous control. Three replicates were used for each treatment. Real‐Time PCR system (Applied Biosystems) was as follows: 95°C for 10 minutes, 40 cycles of 95°C for 10 seconds each and 60.5°C for 30 seconds. The primers used are listed in Table 1. Fold changes in gene expression were calculated by means of relative quantification (2^‐ΔΔCt method).

Total RNA content was extracted from the tissues and cells using TRIzol reagents (16096020, Thermo Fisher Scientific). For mRNA detection, the extracted RNA was reversely transcribed into cDNA using Reverse Transcription Kit (M1701, Promega). The cDNA sample was incubated at 80 °C for 5 min to inactivate the reverse transcriptase for subsequent RT-qPCR detection. For miRNA detection, the extracted RNA was reversely transcribed into cDNA using miRNA First Strand cDNA Synthesis (Tailing Reaction) Kit (B532451, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China). Downstream primers were provided by the kit. RT-qPCR was performed using TaqMan MicroRNA Assay and TaqMan^® Universal PCR Master Mix. GAPDH was regarded as an internal reference of mRNAs while U6 as an internal reference of miRNA. Three replicate wells were set for each sample. The primer sequences are shown in Supplementary Table 2. Relative quantitative method (2^-△△CT method) was used to calculate the relative transcription level of the tested gene [29 (link)].

Total RNA was extracted from cultivated cells. The cDNA of circRNA and mRNA were synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), while the cDNA of miRNA was synthesized using the miRNA First Strand cDNA Synthesis (Tailing Reaction) Kit (Sangon Biotech, Shanghai, China). For quantification, real-time PCRs with the specified primers (Supplementary File S4) were performed using Hieff qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai, China). The levels of mRNA and circRNA were normalized to β-actin, while the levels of miRNA were normalized to U6. Each experiment was repeated 3 times and the expression levels were assessed via the 2^−ΔΔCt method.