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Application of scaRNA9 gene in early judgment of prognosis of acute myeloid leukemia

A technology for acute myeloid and leukemia, applied in the field of leukemia diagnosis

Pending Publication Date: 2021-04-06
北京旌准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are FLT3, IDH1, IDH2 and other genes as markers in the genetic detection period, but each gene cannot give clear information and data during the cell division period to determine the occurrence, development and outcome of future diseases

Method used

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  • Application of scaRNA9 gene in early judgment of prognosis of acute myeloid leukemia
  • Application of scaRNA9 gene in early judgment of prognosis of acute myeloid leukemia
  • Application of scaRNA9 gene in early judgment of prognosis of acute myeloid leukemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 extracts total RNA

[0040] Cell lysis: Add 1mL of QIAZOL reagent to the collected cell pellet, shake and mix well, and let it work at room temperature for more than 15 minutes to fully lyse it. If RNA cannot be extracted immediately after adding QIAZOL reagent, it can be stored at -20°C and can be used in a short time.

[0041] According to the chloroform:QIAZOL volume ratio of 1:4, add about 300 μL of chloroform, mix up and down for 1 minute, let stand at room temperature for 5-10 minutes, and centrifuge at low temperature: 4°C, 12600rpm for 10 minutes.

[0042] After centrifugation, the liquid in the EP tube is divided into three layers, the upper layer is the supernatant containing RNA, and the middle and lower layers are DNA and protein. Then, transfer the supernatant to a new EP tube, add an equal volume of isopropanol, mix up and down, let stand at low temperature for 10 minutes, and centrifuge: 4°C, 12600rpm for 15 minutes.

[0043] Discard the super...

Embodiment 2

[0046] Example 2 RNA reverse transcription

[0047] The reverse transcription system was prepared according to Table 1, and the total reaction volume was 25 μL (total RNA amount was 1 μg). The following operations were performed on ice:

[0048] Table 1 Preparation of reverse transcription system

[0049] components Reaction tube volume (μL) 10nM dNTPs 1.2 reverse transcriptase MMLV 1.3 MMLV RT 5*buffer 5.0 RNAase inhibitor 0.6 random primer 1.0 nuclease free water 15.9 RNA 1μg total 25

[0050] After preparing the above reaction components into 0.2mL PCR reaction tubes, put them into a PCR instrument for reverse transcription reaction, the program is 37°C, 2 hours; 4°C, forever. The product obtained after the reaction is cDNA, and the reaction product is taken out and stored at -20°C until use.

Embodiment 3

[0051] Embodiment 3 real-time fluorescent quantitative PCR detection

[0052] Prepare the RT-PCR mix according to Table 2.

[0053] In the reaction tube, prepare 2 times the volume of 2×SYBR Green I, Nuclease-free water, template cDNA and ROX II mixture, mix well and divide into two 0.2mL PCR reaction tubes, and then add the target gene scaRNA9 respectively The primer for mRNA (SEQ ID NO.1) was paired with SEQ ID NO: 2 and 3 or the primer for internal reference GAPDH (glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase), and mixed gently.

[0054] scaRNA9 upstream primer (SEQ ID NO.2): 5'-TGAGCATCAGAAGTCTTTCCAGTC-3';

[0055] scaRNA9 downstream primer (SEQ ID NO.3): 5'-CACCCTCAATCTCATTCATTCCTAC-3'.

[0056] Table 2 Preparation of RT-PCR mix

[0057] components Reaction tube volume (μL) 2*SYBR Green I 12.5 ROX II 0.5 10μM Primer (F+R) 1.2 cDNA 1.8 Nuclease-free water 9.0 total 25

[0058] Put...

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Abstract

The invention belongs to the technical field of leukemia diagnosis, particularly relates to a detection method and application of a scaRNA9 gene, and particularly relates to application of the scaRNA9 gene as a new marker for AML diagnosis. According to the invention, risk stratification or prognosis evaluation is carried out on patients with acute myelogenous leukemia through the expression level of scaRNA9, and a theoretical basis is provided for the design and selection of personalized medical schemes.

Description

technical field [0001] The invention relates to the technical field of leukemia diagnosis, in particular to the application of scaRNA9 gene in early judgment of prognosis of acute myeloid leukemia. Background technique [0002] Acute myeloid leukemia (AML) is a highly heterogeneous malignant blood disease, accounting for about 80% of acute leukemia in adults. The current standard chemotherapy can achieve about 70-80% complete remission rate (Complete remission, CR), but most of the patients after CR may relapse, and even some patients are difficult to achieve CR and become refractory leukemia, and the treatment fails and dies. One of the main reasons for the highly heterogeneous prognosis of AML is the high genetic heterogeneity of tumor cells. Chromosomal translocation is the most common diagnostic and prognostic marker. In addition, about half of the patients have no chromosomal abnormality, which is called normal karyotype AML (cytogenetically normal AML, CN-AML). [00...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/118C12Q2600/158C12Q2600/178C12Q2531/113C12Q2521/107C12Q2561/113C12Q2563/107
Inventor 叶锋
Owner 北京旌准医疗科技有限公司
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