Marker for early diagnosis, risk stratification and prognosis risk stratification of acute myelogenous leukemia and application thereof
A technique for acute myeloid, early diagnosis, applied in biochemical equipment and methods, microbial determination/examination, DNA/RNA fragments, etc.
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Embodiment 1
[0033] Specific detection primers were designed for the SSBP4 gene, and the SSBP4 gene in cells was amplified by RT-qPCR and sequenced to verify the reliability of the specific detection primers.
[0034] The specific detection primers for the SSBP4 gene include:
[0035] Forward primer: 5'-TACGTTTATGAGTACCTGCTGCACATC-3' (SEQ ID No. 1);
[0036] Reverse primer: 5'-TTCTTCTCCCATCGGATCTCAGACAG-3' (SEQ ID No. 2);
[0037] At the same time, the specific detection primers for the internal reference gene GAPDH were designed, including:
[0038] Forward primer: 5'-ATGGGGAAGGTGAAGGTCG-3' (SEQ ID No. 3);
[0039] Reverse primer: 5'-GGGTCATTGATGGCAACAATATC-3' (SEQ ID No. 4).
[0040] The above two pairs of specific primers were synthesized by Shanghai Sangon Biological Company.
[0041] RT-qPCR amplification methods include:
[0042] (1) Cell collection and pretreatment
[0043] The MV4-11 cell line cultured in vitro was collected, washed twice with PBS, and 1 mL of RNAiso Plus rea...
Embodiment 2
[0059] The above-mentioned specific detection primers are used to detect and analyze the expression of SSBP4 gene in the bone marrow of patients with acute myeloid leukemia. The detection and analysis methods include:
[0060] (1) Collection and processing of bone marrow specimens
[0061] From June 2016 to August 2021, 148 new AML bone marrow samples and 40 normal control bone marrow samples were collected from Ningbo First Hospital. All participants signed the informed consent; bone marrow samples were collected from bone marrow cell culture flasks and recorded. Patient information; mononuclear cells were collected by density gradient centrifugation of lymphocyte separation medium (Ficoll), and finally RNAiso Plus reagent was added to the collected mononuclear cell clumps, repeated pipetting and mixing, and stored at -80°C until use.
[0062] (2) RNA extraction: same as Example 1;
[0063] (3) cDNA reverse transcription: same as Example 1;
[0064] (4) Quantitative PCR: th...
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