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Oligonucleotide, method and kit for detecting relative expression of MLL5 gene in sample

A technology of relative expression and oligonucleotides, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of high cost and poor specificity, and achieve easy operation and predictive The effect of prognosis and improving experimental efficiency

Inactive Publication Date: 2020-01-17
北京艾迪康医学检验实验室有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, because SYBRGreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Oligonucleotide, method and kit for detecting relative expression of MLL5 gene in sample
  • Oligonucleotide, method and kit for detecting relative expression of MLL5 gene in sample
  • Oligonucleotide, method and kit for detecting relative expression of MLL5 gene in sample

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Experimental program
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Effect test

Embodiment 1

[0042] The nucleic acid detection kit used to detect the relative expression of MLL5 in samples can be used to assist in the early prevention, early diagnosis and auxiliary indicators of prognosis of human tumors in clinical practice, and can also perform more accurate screening for high-risk groups. The kit includes: sample extraction reagent; RNA reverse transcription reagent: ReverTra Ace qPCR RT Kit (TOYOBO); detection system PCR reaction solution; positive control, negative control and blank control.

[0043] Wherein, the sample extraction reagent can extract RNA in tissue or blood. Sample extraction reagents and RNA reverse transcription reagents can be prepared by themselves, or purchased from commercial reagents such as related kits from TOYOBO.

[0044] Detection system PCR reaction solution: THNDERBIRD Probe qPCR Mix (2×), MLL5-F, MLL5-R, MLL5-Probe, ABL-F, ABL-R, ABL-Probe; the primer and probe sequences are:

[0045] MLL5-F: CCTTTACCAACACCAGCTACAG

[0046] MLL5-R...

Embodiment 2

[0053] 1. Sample blood total RNA extraction process:

[0054] (1) Collect 1ml of anticoagulated fresh blood, and register age and gender;

[0055] (2) Add 1ml of erythrocyte lysate into a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well. Stand at room temperature for 10 minutes;

[0056] (3) Centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom;

[0057] (4) Add 0.5ml red blood cell lysate again, centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom;

[0058] (5) Add 0.2ml TRIzol to the cells, pipette repeatedly until the precipitate is completely dissolved, mix 5 tubes of TRIzol digestion solution into 1 tube, and let stand at room temperature for 5 minutes;

[0059] (6) Add 0.2ml chloroform and shake evenly;

[0060] (7) Centrifuge at 14000rpm at 4°C for 10min, absorb the supernatant layer and transfer it to another new centrifuge tube (do not absorb the white middle...

Embodiment 4

[0077] The detection kit of the invention is used to detect normal people and blood samples.

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Abstract

The invention provides a method, a kit and an oligonucleotide for detecting relative expression of the MLL5 gene in a sample, and all relate to an upstream primer MLL5-F for detection, an upstream primer MLL5-R for detection and a probe MLL5-Probe for detection, and an upstream primer ABL-F for the internal reference gene ABL, a downstream primer ABL-R and a probe ABL-Probe. The relative expression of the MLL5 gene in clinical samples can be rapidly detected. The invention can be applied to evaluation of therapeutic effect and prediction of prognosis.

Description

technical field [0001] The invention belongs to the field of biological science and biotechnology, and particularly relates to a method for detecting the relative expression level of MLL5 gene in a sample, an oligonucleotide and a kit, which are used to detect the expression level of MLL5 gene in tumor patients by adopting fluorescent PCR technology. Background technique [0002] Acute myeloid leukemia (AML) is a heterogeneous disease characterized by distinct recurrent cytogenetic abnormalities that provide the most important prognostic information for diagnosis. In recent years, different mutations in multiple genes such as FLT3, NPM1, CEBPA, IDH and WT1 have been found in AML. Mutations in some of these genes are associated with treatment outcome in cytogenetically normal AML and thus serve as the basis for molecularly directed risk assessment and stratification of therapy. In addition, the normal regulation of the expression of genes important for proliferation, surviva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2561/101
Inventor 刘赵玲李允章王淑一
Owner 北京艾迪康医学检验实验室有限公司
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