Trizol lysate

The expression levels of LncRNA H19 in peripheral blood of the two groups were detected by qRT-PCR, the total RNA was extracted from lytic cells with TRIzol lysate (Thermo Fisher Scientific (China)), 15596018), procedures of extraction followed the instructions in the kit. The concentration and purity of the extracted RNA were analyzed by Evolution™ 201/220 ultraviolet spectrophotometer (Thermo Fisher Scientific (China)), and the A260/A280 values between 1.8 and 2.1 were considered to meet the experimental requirements. Three percent agarose gel electrophoresis (the gel electrophoresis kit was purchased from Shanghai jingke chemical technology Co., Ltd.) was used to analyze the integrity of RNA. EasyScript One-Step RT-PCR SuperMix kit was purchased from Beijing Transgen Biotech, RRNA Template 1 μg, Forward GSP (10 μM) 0.4 μl, Reverse GSP(10 μM) 0.4 μl, 2*One-Step Reaction Mix 10 μl, EasyScript One-Step Enzyme Mix 0.4 μl, rnrnase-free Water supplementary Reaction system value 20 μM, the reaction conditions were 40 °C 30 min, 94 °C 5 min, 94 °C 30 s, 60 °C 30 s, 72 °C 2 kb/min and 72 °C 10 min with a total of 40 cycles. The β-actin was used as the internal reference of the reaction, 2−ΔCt was used to analyze the results. Primer sequences were designed and synthesized by Thermo Fisher Scientific (China) (Table 1).

In order to verify the expression levels of each lncRNA of CRLncSig, nine pairs of LUAD tissues and adjacent normal tissues were examined using real-time PCR. The Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University approved this study. Informed consent was obtained from all patients before surgery, no history of chemotherapy or radiotherapy was present before surgery. After extracting the tissues, we froze and stored them in liquid nitrogen. Total RNA was extracted from the sample tissues via Trizol lysate (Thermo Fisher Scientific). We reverse-transcribed total RNA from clinical samples into cDNA using the HiScript III RT SuperMix Kit (R32301, Vazyme, Nanjing, China). The real-time PCR was performed using the CFX96 system (BIO-RAD Laboratories, Inc., Hercules, CA, United States). Based on the 2^−ΔΔCT method, expression levels of target RNAs were normalized to GAPDH. The mean value was used as the final experimental result for replicated wells. The manufacturer’s instructions were followed during all procedures. Student’s t-test was applied to identify genes differentially expressed between normal and tumor samples. Differentially expressed genes were defined as adjusted p-value < 0.05.

Total RNA was extracted from mouse liver and cell samples using Trizol lysate (Thermo Fisher Scientific), and then reversed to cDNA using Hifair II 1st Strand cDNA Synthesis Kit (Shanghai Yeasen Biotech). The real-time PCR system was 20 μl in total which included 10 μl SYBR PCR mix, 2 μl cDNA template, 0.4 μl sense primers and anti-sense primers (The primer information is presented in Table 1) and 6.8 μl deionized water. PCR reaction programme was set as follows: pre-denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s; collection of melting curves at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. β-actin was used as an internal reference to correct the relative expression of genes, and 2^−ΔΔCt method was used for statistical analysis of gene expression level. GraphPad Prism 5 software was used to analyze the level of data significance among different groups.

qRT-PCR was performed on 10 pairs of RC tissues and adjacent normal tissues to validate the mRNA expression levels of the 14 signature genes. Consent forms were obtained from each patient for the collection and analysis of tissue samples. The study was approved by the Peking Union Medical College Hospital ethics review boards. We immediately froze and stored the tissues in liquid nitrogen after extracting them. Total RNA was extracted from the sample tissues via Trizol lysate (Thermo Fisher Scientific), followed by reverse transcription to cDNA. qRT-PCR was carried out using the CFX96 system (BIO-RAD CFX96; BIO-RAD Laboratories, Inc., Hercules, CA, USA). GAPDH served as an internal control. Relative expression levels were quantified by the Ct (2^−ΔΔCt) method, and the mean value was used as the final experimental result for replicated wells. All procedures were carried out in accordance with the manufacturer’s instructions.

RNA was extracted, and endometrial cancer tissues and paracancerous tissues were ground into powder in liquid nitrogen. In every 30 mg of tissue powder and HEC-1B cells and radiation-resistant HEC-1B cells, 1 mL of TRIzol lysate (Invitrogen, Carlsbad, CA, USA) was added for lysis, then transferred to a centrifuge tube. Then, the mixture was evenly mixed and centrifuged at a radius of 9 cm and a speed of 12,000 r/min for 15 min. After that, 75% ethanol was added to wash and dry. The concentration of RNA was detected by ultraviolet spectrophotometer, and the level of BMI1 mRNA was detected by qRT-PCR kit. The upstream of BMI1 mRNA was 5′-ATGATAAAAGATACTTACGATGCCCAG-3′, and the downstream was 5′-GAACTCTGTATTTCAATGGAAGTGGAC-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. The upstream sequence of the primer was 5′-AACAGGCGTGGCGGCTTCTACCA-3′, and the downstream sequence was 5′-GCTGCCTTGGTTGAGAAAGT-3′. The PCR amplification was performed in a 20 μL reaction under the condition of 95°C, 5 min, with 40 cycles of denaturation for 40 s, annealing at 72°C for 30 s, extension at 62°C for 10 s. The level of BMI1 mRNA in tissues was calculated by relative quantitative 2^−ΔΔCT.

TRIzol lysate was purchased from the Invitrogen Company (Frederick, USA), The revertAid first stand cDNA synthesis kit was purchased from the Thermo Company (Lithuania, EU), the Fluorescence quantitative RT-PCR kit was purchased from the Takara Company (Dalian, China), the HO-1 primers were synthesized by the Invitrogen Company (Shanghai, China), the anti-heme oxygenase 1 antibody was purchased from the Abcam Company (Cambridge, MA, USA), the immunohistochemistry kit was purchased from the Invitrogen Company (Frederick, USA).