Sybr green mastermixs kit

Lentivirus transfected cells EJ, T24, J82, and RT4 were cultured for 72 h, and total RNA was extracted using TRIzol reagent (Sigma, St. Louis, MO, USA), respectively. Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the quality of RNA according to the manufacturer’s instructions. Then 2.0 μg RNA sample was reverse transcribed to cDNA and quantitative real-time PCR was conducted with SYBR Green mastermixs Kit (Vazyme, Nangjing, Jiangsu, China) and 2^−ΔΔCt method was applied to evaluate the relative quantitative. Inner control of PCR was GAPDH. Primers used were showed in Table S2.

To extract total RNA, TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) was utilized. Subsequent to isolating DNA, cDNA synthesis and qRT-PCR were conducted on cholangiocarcinoma cell lines transfected with indicated lentivirus, employing the Promega M-MLV Kit (Promega Corporation, Madison, WI, USA) and the SYBR Green Mastermixs Kit (Vazyme, Nanjing, China). GAPDH served as an internal normalization control, and the 2^-∆∆Ct method was employed to evaluate relative mRNA expression. The primers sequences (5′-3′) were listed in Supplementary Table 2.

After 72 h for ZNF280A and/or EIF3C RNA expressing, A549 and NCI-H1299 cells in triplicate were fully lysed and total RNA was extracted using TRIzol reagent (Sigma). The RNA quality was evaluated by Nanodrop 2000/2000C spectrophotometer (Thermo Fisher Scientific). cDNA was reversely transcribed from RNA using Promega M-MLV Kit (Promega) and qPCR was performed with SYBR Green mastermixs Kit (Vazyme) by applying Biosystems 7500 Sequence Detection system. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was acted as inner control, and the primers used for the PCR reaction were showed in Table S3. The relative quantitative analysis in gene expression data were analyzed by the 2^−ΔΔCt method.

Total RNA was extracted according to TRIzol reagent (Sigma, St. Louis, MO, USA). The concentration and quality of the total RNA were determined by Nanodrop 2000/2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, the reverse transcription was performed to obtain cDNA by using the Promega M-MLV Kit (Promega Corporation, Madison, Wisconsin, USA). The qRT-PCR system was 10 μL according to the SYBR Green Mastermixs Kit (Vazyme, Nangjing, Jiangsu, China). GAPDH was served as internal reference. The relative expression level was calculated based on the 2^−△△Ct method. The primers sequences (5′–3′) were presented in Supplementary Table 2.

Eca-109 and TE-1 cells were lysed and total RNA was isolated using TRIzol reagent (Thermo, USA). RNA concentration was determined using Nanodrop 2000/2000C spectrometer (Thermo, USA). The cDNA was reversely transcribed from RNA using M-MLV Kit (Promega, USA) and qRT-PCR was performed with SYBR Green mastermixs Kit (Vazyme) and Biosystems 7500 Sequence Detection system. GAPDH was used as inner control, and the primers used for the PCR reaction were shown in Additional file 1: Table S3. The relative quantitative data of gene expression were analyzed by the 2^−ΔΔCt method.

Total RNA from shRNA expressing lentivirus infected cells MEC-2 and M01043 were extracted using TRIzol reagent (Sigma). After quantification by a Nanodrop 2000/2000 C spectrophotometer (Thermo Fisher Scientific), 500 ng of RNA was used for cDNA synthesis. RT-PCR was performed with a Biosystems 7500 Sequence Detection system using a SYBR Green Mastermixs Kit (Vazyme). Relative gene expression was analyzed by the 2^−ΔΔCt method. Reactions were performed in triplicate and the corresponding primers used in PCR are detailed in Table S4.

The cells infected lentivirus were collected and centrifuged at 2000 rpm for 5 min, then 1 mL Trizol was added for RNA extraction according to the manufacturer’s instruction of TRIzol reagent (Sigma, St. Louis, MO, USA). cDNA was obtained by reverse transcription according to the instructions of Promega M-MLV Kit (Promega Corporation, Madison, Wisconsin, USA). Real-time quantitative PCR (qRT-PCR) was performed based on the steps of SYBR Green Mastermixs Kit (Vazyme, Nanjing, Jiangsu, China). The qRT-PCR reaction volume was 10 μL, and the relative expression level of RNA was calculated by the 2^−△△Ct method. GAPDH was used as an internal control.
The primers sequences used in qPCR were as follows (5′–3′): The forward primer of RPL35A was 5′-GAAGGTGTTTACGCCCGAGAT-3′, the reverse primer of RPL35A was 5′-CGAGTTACTTTTCCCCAGATGAC-3′. The forward primer of GAPDH was 5′-TGACTTCAACAGCGACACCCA-3′, the reverse primer of GAPDH was 5′-CACCCTGTTGCTGTAGCCAAA-3′.