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Annular RNA circBA9.3 and use thereof to preparation of CML diagnosis reagent kit

A BCR-ABL1 and kit technology, applied in the field of CML diagnostic kits, can solve the problems of low circular RNA expression, difficulty in detection, lack of internal reference genes, etc.

Inactive Publication Date: 2019-05-03
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression level of most circular RNAs is extremely low, which is difficult to detect by conventional PCR methods, and there is a lack of internal reference genes to accurately measure the expression level of circular RNAs in vivo

Method used

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  • Annular RNA circBA9.3 and use thereof to preparation of CML diagnosis reagent kit
  • Annular RNA circBA9.3 and use thereof to preparation of CML diagnosis reagent kit
  • Annular RNA circBA9.3 and use thereof to preparation of CML diagnosis reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: The circular sequence of circBA9.3 was amplified from a mononuclear cell sample of a BCR-ABL1 positive CML patient. The mononuclear cell sample was derived from the peripheral blood of a CML patient and collected by the Department of Hematology, Shenzhen Second People's Hospital , the patient has signed the informed consent. The time of collection is different for different patients, and the approximate collection time of samples is 2016.01-2017.12.

[0049] (1) RNA extraction and DNA digestion

[0050] 1) Take 1ml of peripheral blood sample from a CML patient, add 3ml of erythrocyte lysate, shake gently at 4°C for 15min, centrifuge at 450g for 10min, and remove the supernatant. Add 2ml of erythrocyte lysate, shake and mix for 5min, centrifuge at 450g for 10min, and remove the supernatant. Add 1×PBS to suspend cells, take 1×10 6 cells, and centrifuged to pellet the cells.

[0051] 2) Add 1ml Trizol reagent, shake and mix for 20s, and let stand at room tem...

Embodiment 2

[0073] Example 2: Construction of CML cell line stably transformed by overexpressing circular RNA circBA9.3

[0074] (1) Construction of overexpression circBA9.3 lentiviral vector

[0075] The circular sequence of circBA9.3 was chemically synthesized, and the sequence was annealed to form a double-stranded DNA fragment, which was inserted into the lentiviral vector LV-Circ expressing circular RNA. Positive clones of recombinant plasmids were identified by sequencing.

[0076] (2) Packaging of lentivirus

[0077] 1) Prepare pSPAX2, pMD2G and LV-Circ-circBA9.3, the plasmid concentration is greater than 1 μg / μL, and the quality A260 / 280 is between 1.7 and 1.8 for transfection.

[0078] 2) The 293T cells in the logarithmic growth phase were transfected when the density reached 70-80% confluence. Add fresh complete culture solution DMEM+10% FBS+1% double antibody before transfection.

[0079] 3) Preparation of transfection system in each well of 12-well plate: 1.6ug pSPAX2, 0.8...

Embodiment 3

[0088] Example 3: Establishment of a 3D digital PCR system for detecting circBA9.3.

[0089] (1) RNA extraction and DNA digestion of overexpressed circBA9.3 cells

[0090] Collect 1×10 by centrifugation 6 cells, 600rpm, 5min. Add 1ml trizol reagent, extract RNA as in Example 1, and digest DNA.

[0091] (2) Get 4ug RNA reverse transcription into cDNA, the steps are the same as in Example 1.

[0092] (3) Dilute the cDNA template one-half times, and set 6 concentration gradients.

[0093] (4) 3D digital PCR reaction system: 2×digital PCR Mix 7.25ul, upstream and downstream primers 0.4ul each, probe 0.3ul, cDNA template 1ul, add water to make up to 14.5ul.

[0094] (5) The reaction conditions of 3D digital PCR are: 96°C for 10min; 60°C for 2min, 98°C for 30s, a total of 39 cycles; 60°C for 2min; storage at 10°C.

[0095] (6) use https: / / china.apps.thermofisher.com / quantstudio3d / online site, to obtain the copy number of circBA9.3. According to different template concentrat...

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Abstract

The invention discloses annular RNA circBA9.3 and use thereof to preparation of a CML diagnosis reagent kit. The annular RNA circBA9.3 is from annular mRNA of a BCR-ABL1 fusion gene, wherein the 3rd exon of 3' end ABL1 is backwards spliced to the 9th exon of 5' end BCR to form an end-to-end annular structure, and the nucleotide sequence of the annular RNA circBA9.3 is as shown in SEQID NO:8. The reagent kit can specifically detect the expression level of circBA9.3 in clinical samples of chronic medullocell leukaemia (CML), so that CML patients can be researched, and a diagnosis and treatment method can be provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CML diagnostic kit. Background technique [0002] The BCR-ABL1 fusion gene is located on the Philadelphia chromosome (Ph) formed by the reciprocal translocation of chromosomes 22 and 9. This fusion gene is an important factor for the diagnosis, prognosis and recurrence prediction of patients with chronic myelocytic leukemia (CML). Marker molecules. The etiology of CML is mainly due to the sustained tyrosine kinase activity of BCR-ABL1, which promotes cell proliferation and anti-apoptosis. Tyrosine kinase inhibitors (TKIs) are competitive inhibitors of the binding of adenosine triphosphate (ATP) to tyrosine kinases, and are clinically used as targeted drugs for the treatment of CML patients. The resistance to TKIs in patients with CML in the chronic phase often leads to the progression of the disease to the accelerated phase and the blast phase, which brings difficulties to the tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6886C12Q1/6851
Inventor 张巧霞杜新潘昱名楼瑾陈欢安娜
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN