A detection and application of crna-fut8 for hepatocellular carcinoma screening
A technology for hepatocellular carcinoma and uses, applied in the field of circular RNA, can solve the problems of no treatment method and limited surgical efficacy, and achieve the effects of high sensitivity, good specificity and good application prospects
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Embodiment 1
[0046] Example 1: Analysis of cRNA-FUT8 chip expression in human hepatocellular carcinoma and paracancerous tissues
[0047] 1. Materials and methods
[0048] 1. Materials
[0049] Tissue samples were obtained from surgical resection samples of 3 pairs of inpatients with HCC (all tissue samples were obtained from the Department of Liver Tumor Surgery, Shanghai Zhongshan Hospital), and each pair contained HCC tissue and paired adjacent normal tissue.
[0050] 2. Method
[0051] (1) Extraction of total RNA from tumor tissue and normal tissue: Total RNA from hepatocellular carcinoma tissue and paracancerous tissue was extracted according to the instructions of Qiagen's RNA extraction kit (RNeasy Micro Kit, Cat. No. 74004).
[0052] (2) Cy5 fluorescent labeling of sample RNA (Shanghai Kangcheng Bioengineering Co., Ltd. is entrusted with "ArrayStar Human cRNA-FUT8 Microarray V3.0 Service" for labeling service)
[0053] (3) Synthesis of first-strand cDNA by reverse transcription:...
Embodiment 2
[0061] Example 2: qRT-PCR preliminary verification of differential expression of cRNA-FUT8 in cancer tissues and paracancerous tissues of hepatocellular carcinoma
[0062] 1. Experimental materials
[0063] 28 pairs (different from the samples tested by the microarray) of human hepatocellular carcinoma tissues (provided by the Department of Liver Tumor Surgery, Shanghai Zhongshan Hospital) and paired paracancerous tissues were selected to verify the expression difference of cRNA-FUT8 by qRT-PCR.
[0064] 2. Experimental methods and results
[0065] 1. Identification of primer specificity
[0066] (1) Design of specific primers: extract cRNA-FUT8-related transcript sequences from the Ensemble database, and use the primer design tool (Primer BLAST) of NCBI to design primers according to the sequence of the transcript;
[0067] The designed primer sequences are as follows:
[0068] Upstream primer: SEQ ID No.2
[0069] Downstream primer: SEQ ID No.3
[0070] (2) Total RNA wa...
Embodiment 3
[0109] Example 3: Using differential expression of cRNA-FUT8 to screen hepatocellular carcinoma tissues
[0110] 1. Experimental materials
[0111] 100 human hepatocellular carcinoma tissues and 100 para-cancerous tissues (provided by Shanghai Renji Hospital) were selected to detect the expression difference of cRNA-FUT8 by qRT-PCR.
[0112] 2. Experimental methods and results
[0113] 1. Identification of primer specificity
[0114] (1) Use the following specific primer sequences:
[0115] Upstream primer: SEQ ID No.2
[0116] Downstream primer: SEQ ID No.3
[0117] (2) Total RNA was extracted from human hepatocellular carcinoma tissues and paracancerous tissues according to the reagents and steps required by SIGMA's TRIZOL reagent (Product No. T9424), and then quantified by 7300 real time PCR system nucleic acid quantification instrument (Applied Biosystems AB). Purity and concentration of RNA.
[0118] (3) Using StarScript II One-step RT-PCR Kit (Product No. A215-01) ...
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