Specific miRNA related to M5 acute myeloid leukemia and its application
An acute myeloid and leukemia technology, applied in specific miRNA and its application fields, can solve the problems of single detection method, affecting the accuracy of diagnosis, delaying the disease, etc., and achieve the effect of broad application prospects and good clinical application value
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Embodiment 1
[0038] Example 1 Screening of highly expressed miRNAs in M5 acute myeloid leukemia cell lines
[0039] By analyzing and comparing M5 leukemia cell lines (THP-1, THP-1 derived from patients with acute monocytic leukemia) and non-M5 myeloid leukemia cell lines (K562, K562 derived from patients with chronic myeloid leukemia) Through-put miRNA-seq data (NCBI accession number: GSE48059) screened 16 highly expressed miRNAs in M5 AML cell lines.
[0040] The above-mentioned 16 miRNA-specific primer pairs were designed, and the expression levels of each miRNA were verified by ABI 7500 fluorescent quantitative PCR instrument, using the RNA tailed reverse transcription products of M5 type THP-1 cell line and non-M5 type K562 cell line as templates. The following table lists 16 miRNAs and their specific amplification primers.
[0041] Table 1
[0042]
[0043]
[0044] Comparing M5 type acute myeloid leukemia cell line (THP-1) and non-M5 type chronic myeloid leukemia cell line (K...
Embodiment 2
[0048] Example 2 Establishment of RT-PCR detection method for M5 acute myeloid leukemia
[0049] 1. Sample collection: 12 bone marrow samples from M5 AML patients and 1 normal bone marrow sample.
[0050] 2. Sample processing: extract total RNA according to the standard operation of Trizol reagent.
[0051] 3. Sample detection: Take 2 μg of total RNA.
[0052] (1) RNA tailing Mix the ingredients in Table 2 in a 0.2mL PCR tube, then bathe in water at 37°C for 1.5 hours.
[0053] Table 3 RNA tailing reaction system.
[0054] Reagent
volume
RNA (DNA free)
2μg
10×Poly(A)Polymerase Reaction Buffer
1μL
ATP (10mM)
1μL
E.coli Poly(A) Polymerase (5U / μl)
0.2uL
Add decalculated water to make up to
20 μL
[0055] (2) Add reverse transcription primers: add 0.5 μl (1 μg / μl) reverse transcription primers, place in a 65-degree water bath for 10 minutes, then place on ice for at least 5 minutes, and keep on ice until R...
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