Marker for prognosis of liver cancer
A liver cancer prognosis and marker technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve unclear problems, achieve accurate evaluation, high clinical value, and significant sexual effect
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Embodiment 1
[0103] Example 1: Extraction of RNA and synthesis of cDNA
[0104] Liver cancer tissues and adjacent normal tissues were obtained from 120 liver cancer patients who had been diagnosed as having developed liver cancer and whose development had been confirmed. RNA was extracted from each tissue and cDNA was synthesized according to the following method.
[0105] Total RNA was extracted from liver cancer tissues and adjacent normal tissues using RNeasy Minikit (Qiagen, Germany) according to the product instructions. The total RNA in the obtained RNA extract was weighed using Bioanalyzer 2100 (Agilent Technologies, USA). DNase I treatment was performed during the extraction step to remove contaminating genomic DNA from the RNA extract. A sample containing 4 μg of total RNA was incubated with 2 μl of 1 μM d(T)18 primer (Genotech, Korea) at 70° C. for 7 minutes and cooled on ice for 5 minutes. Prepare enzyme mix separately [by adding 2 μl of 0.1 M DTT (Duchefa, Netherlands), 2 μl...
Embodiment 2
[0106] Example 2: Quantitative real-time RCR
[0107] According to the product manual, use PRISM 7900HT (Applied Biosystems, USA) to implement quantitative real-time RCR amplification for the following two gene markers of each cDNA sample obtained in Example 1:
[0108] CBS (cystathionine β-synthase; NCBI GI: 209862802; SEQ ID NO: 79); and
[0109] NNMT (nicotinamide N-methyltransferase; NCBI GI: 62953139; SEQ ID NO: 80).
[0110]The quantitative real-time RCR analysis was carried out in a total volume of 10 μl, which included 5 μl of 2×Taqman gene expression master primer (master mix) (Applied Biosystems, USA), 1 μl each of 5 μM forward and reverse primers, 1 μl 1 μM probe (Genotech, Korea) and 2 μl of cDNA (equal amount of water in the control group). Amplification was performed in the following cycle: a 95°C dissociation step for 10 minutes, followed by a 95°C dissociation step for 15 seconds, and a 60°C synthesis step for 1 minute. Primer and probe sequences were design...
Embodiment 3
[0117] Example 3: Data Analysis
[0118] Considering the normalized expression of each marker obtained in Example 2 and the progression of patients providing liver cancer tissues, Kaplan-Meier curves were completed, followed by significance analysis.
[0119] According to the progression of 120 patients who provided liver cancer tissue, the patients are listed in order of ascending stage for each case of recurrence, overall survival and disease-free survival. The interval survival rate (or interval recurrence rate) was calculated by dividing the number of survivors (or relapsed patients) by the number of patients exposed to risk. The cumulative survival rate (or cumulative recurrence rate) is a conditional probability calculated by multiplying the previous cumulative survival rate (or cumulative recurrence rate) by the present median recurrence rate (median recurrence rate). With the survival time (or observation period) as the horizontal axis and the cumulative survival rate...
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