A preparation for early diagnosis of patients with chronic heart failure including serum long non-coding RNA H19
A chronic heart failure, long-chain non-coding technology, applied in the measurement/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of high morbidity and mortality, and achieve far-reaching clinical significance and promotion value. High sensitivity and good stability
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Embodiment 1
[0023] Real-time fluorescent quantitative PCR detection found that the expression of lncRNAH19 was increased in myocardial tissue of patients with chronic heart failure.
[0024] 1. Materials and methods
[0025] Total RNA was extracted from 6 cases of chronic heart failure patients and 7 cases of normal human myocardial tissue, 1 μg of RNA was reverse transcribed into cDNA, and then detected by real-time fluorescent quantitative PCR.
[0026] The sequence of the serum lncRNA H19 forward primer is shown in SEQ NO.2: 5'-TCCCAGAACCCACAACATGAAA-3'.
[0027] The sequence of the serum lncRNA H19 reverse primer is shown in SEQ NO.3: 5'-CACCTTCCAGAGCCGATTCC-3'.
[0028] The sequence of the U6 snRNA forward primer used for internal reference is shown in SEQ NO.4: 5'-ATTGGAACGATACAGAGAAGATT-3'.
[0029] The sequence of the U6 snRNA reverse primer used for internal reference is shown in SEQ NO.5: 5'-GGAACGCTTCACGAATTTG-3'.
[0030] The real-time fluorescence quantitative PCR reaction...
Embodiment 2
[0037] Real-time fluorescence quantitative PCR method detection found that the expression of serum lncRNAH19 was significantly increased in patients with chronic heart failure.
[0038] 1. Materials and methods
[0039] The pre-collected peripheral blood serum samples from the above-mentioned 6 patients with chronic heart failure and 7 normal subjects were placed on ice for future use. The serum collection process is as follows. Use BD vacuum blood collection tubes (heparin anticoagulant) to collect about 5 mL of fasting venous blood from patients with chronic heart failure and normal people. After standing at room temperature for 30 minutes, centrifuge at 4°C and 1600g for 10 minutes. Carefully transfer and aliquot the upper serum into new 1.5mL centrifuge tubes, and freeze at -80°C for later use.
[0040] Utilize QIAamp Circulating Nucleic Acid Kit (Qiagen, #55114) (this kit is a commercial RNA extraction kit, produced by Qiagen Company) kit to extract total RNA in serum, t...
Embodiment 3
[0066] By expanding the sample size, the specificity and sensitivity of real-time fluorescent quantitative PCR to detect inflammatory serum lncRNA H19 for the diagnosis of chronic heart failure.
[0067] 1. Materials and methods
[0068] About 5 mL of fasting venous blood was collected from 100 patients with chronic heart failure and 100 normal subjects using BD vacuum blood collection tubes (heparin anticoagulant). After standing at room temperature for 30 minutes, they were centrifuged at 4°C and 1600g for 10 minutes. Carefully transfer and aliquot the upper serum into new 1.5mL centrifuge tubes, and freeze at -80°C for later use.
[0069] Using the steps in Example 2, total RNA in serum was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, #55114) (this kit is a commercial RNA extraction kit, produced by Qiagen).
[0070] The steps in Example 2 were used to detect the expression of lncRNA H19 by reverse transcription and real-time fluorescence quantification...
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