Prostate cancer early diagnosis or prognosis evaluation marker lncRNA malat1 and application thereof
A technology for prostate cancer and prognosis evaluation, applied in the field of medical diagnosis, can solve the problems of early diagnosis of unexposed prostate cancer, undisclosed relationship between prostate cancer, etc., and achieves the effect of high sensitivity, specificity, and good indication effect.
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[0041] Example 1
[0042] The main purpose of this example is to detect the content of each malat1 fragment of serum exosomal-derived malat1, which specifically includes:
[0043] 1. Use the exosome extraction kit (QIAGEN company: exoEasy Maxi Kit or miRCURY ExosomeKits; Thermofisher company: Total Exosome Isolation Reagent from serum) to extract the total exosomes in the serum. Using the ribonucleic acid extraction kit (QIAGEN company: miRNeasyMicro Kit), the extracted exosomes were used to extract all the ribonucleic acids in the exosomes. The extracted ribonucleic acid is converted into cDNA using a reverse transcription kit (Takara: PrimeScript™ II 1st Strand cDNA Synthesis Kit).
[0044] 2. Design primers to amplify different fragments on malat1 based on the nucleotide sequence of malat1. The specific primer information is shown in Table 1. Use the primers shown in Table 1 and perform PCR reaction and agarose gel electrophoresis on the cDNA (see electrophoresis results figure ...
Example Embodiment
[0047] Example 2
[0048] The main purpose of this embodiment is to establish a method for quantitative detection of exosomal malat1-1 fragments, and the method includes:
[0049] 1. Extract serum total exosomes and ribonucleic acid, and reverse transcribe the ribonucleic acid into cDNA (see Example 2 for specific methods).
[0050] 2. According to the sequence of malat1-1, a probe designed for use with the upstream and downstream primers of malat1-1: wherein the nucleotide sequence of the malat1-1 is:
[0051] TAGGGGATTTCAGGATTGAGAAATTTTTCCATCGAGCCTTTTTAAAATTGTAGGACTTGT (SEQ ID NO: 13)
[0052] The nucleotide sequence of the probe is: TCCATCGAGCCTTTT (SEQ ID NO: 14), the 5'end of the probe is labeled with FAM fluorescent group, and the 3'end is labeled with MGB modification group.
[0053] 3. A nucleic acid fragment of artificially synthesized malat1 is used as a nucleic acid standard and diluted to concentrations of 5pg / μL, 1pg / μL, 0.5pg / μL, 0.2pg / μL, 0.1pg / μL. The artificially synthes...
Example Embodiment
[0056] Example 3
[0057] The main purpose of this example is to evaluate the diagnostic effect of serum exosomal-derived malat1-1 on early prostate cancer, including:
[0058] 1. Convene 299 subjects whose clinical diagnosis results have been confirmed. See Table 2 for specific information.
[0059] 2. Detect the serum exosome-derived malat1 of 299 subjects (see Example 2 for the detection method) and plasma MD-miniRNA (see the Chinese invention patent CN103146688B for the detection method).
[0060] 3. According to the subject's diagnosis results, the copy number of serum exosomal-derived malat1, and plasma MD-miniRNA concentration, the ROC curves of exosome-derived malat1 and MD-miniRNA were drawn by SPSS13.0 software ( See image 3 ).
[0061] 4. Conclusion: According to image 3 The results show that the AUC area (0.881), sensitivity and specificity of the ROC curve of serum exosomal malat1 are higher than the existing marker MD-miniRNA (AUC area is 0.756), indicating that the mar...
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