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LTL (leukocyte telomere length) detecting method based on qPCR (quantitative polymerase chain reaction) method

A technology of telomere length and white blood cells, applied in the field of molecular genetics, can solve the problems of cumbersome operation, unfavorable technology popularization and application, long time consumption, etc., and achieve the effects of high reliability of results and simple and feasible experimental operation process.

Inactive Publication Date: 2017-03-08
浙江指针基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Scientists have been studying the determination of telomere length since the 1980s, and have successively established a series of methods for detecting telomere length, such as FISH, Southern Blot, Flow-FISH, etc. Among these methods, Southern Blot is more intuitive, but The required genome is large (>1ug), and the operation is cumbersome and time-consuming
Although a series of detection methods using the FISH platform are fast and convenient, they have higher requirements for samples.
Not conducive to the promotion and application of technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A method for detecting leukocyte telomere length based on qPCR method of the present embodiment comprises the following steps:

[0026] 1) Extract genomic DNA from different tissues, dilute the DNA sample concentration to 5ng / ul, take 5ul of each tissue sample and combine and mix to make a whole-genome DNA template;

[0027] Extraction of whole-genome DNA: Genome DNA comes from blood, tissue, saliva, sperm or eggs. The specific steps for extracting whole-genome DNA by phenol / chloroform method are as follows:

[0028] (1) Thaw and mix the frozen anticoagulated blood at room temperature;

[0029] (2) Take 200ul of fresh blood and put it into a 1.5ml centrifuge tube;

[0030] (3) Add 200ul cell lysate, 30ul Proteinase K, mix with vortex for 15s, and bathe in water at 55°C for 10min;

[0031] (4) Add an equal volume of phenol, about 500ul, vortex for 15s, and centrifuge at 12000rpm for 5min;

[0032] (5) Take the supernatant, add an equal volume of phenol / chloroform / isoa...

Embodiment 2

[0047] A method for detecting leukocyte telomere length based on qPCR method of the present embodiment comprises the following steps:

[0048] 1) Extract genomic DNA from different tissues, dilute the DNA sample concentration to 5ng / ul, take 5ul of each tissue sample and combine and mix to make a whole-genome DNA template;

[0049] Extraction of whole-genome DNA: Genome DNA comes from blood, tissue, saliva, sperm or eggs. The specific steps for extracting whole-genome DNA with the QuickGene610L automatic extractor are as follows:

[0050] 1) Thaw and mix the frozen anticoagulated blood at room temperature;

[0051] 2) Take a new 15ml centrifuge tube, add 300ml EDB solution, 2ml whole blood, and 2.5ml LDB solution in sequence;

[0052] 3) Mix upside down, vortex for 15 seconds, and water bath at 55°C for 10 minutes;

[0053] 4) Add 2.5ml of absolute ethanol, mix well, vortex for 15s;

[0054] 5) Pour the processed whole blood into the QuickGene 610L filmed sleeve;

[0055] ...

Embodiment 3

[0067] A method for detecting leukocyte telomere length based on qPCR method of the present embodiment comprises the following steps:

[0068] 1) Extract genomic DNA from different tissues, dilute the DNA sample concentration to 5ng / ul, take 5ul of each tissue sample and combine and mix to make a whole-genome DNA template;

[0069] Extraction of whole-genome DNA: Genome DNA comes from blood, tissue, saliva, sperm or eggs. The specific steps for extracting whole-genome DNA using the QIAamp DNAMini Kit kit are as follows:

[0070] 1) Take 200ul of fresh blood and place it in a 1.5ml centrifuge tube;

[0071] 2) Add 200ul cell lysate, 30ul Proteinase K, mix well with vortex for 15s, and bathe in water at 55°C for 10min;

[0072] 3) Add 200ul of absolute ethanol to the centrifuge tube, mix with vortex for 15s;

[0073] 4) Transfer the liquid in the centrifuge tube to a new QIAamp Mini spin column;

[0074] 5) Centrifuge at 8000rpm for 1min, discard the liquid, and add 500ul Buf...

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Abstract

The invention discloses an LTL (leukocyte telomere length) detecting method based on a qPCR (quantitative polymerase chain reaction) method. The method comprises steps as follows: 1) extracting genomic DNA of different tissue, diluting DNA samples to 5 ng / ul, taking 5 ul of each tissue sample, and mixing the tissue samples to prepare a whole genomic DNA template; 2) performing primary real-time qPCR detection and detecting a telomere primer of a telomere repetitive sequence; 3) performing secondary real-time qPCR detection and transcribing a single-copy reference RNase P gene of RNase P enzyme; 4) calculating the relative telomere length T / S. The LTL detecting method based on the qPCR method has the benefits as follows: the requirement for the samples and genomic DNA concentration is not high, the experiment operation process is simple and feasible, the result credibility is high, and the method is adaptable to scientific research and clinical popularization and application and plays an important role in preventing aging, cancer and diseases.

Description

technical field [0001] The invention relates to the field of molecular genetics, in particular to a method for detecting leukocyte telomere length based on a qPCR method. Background technique [0002] Telomere (Telomeres) located at the end of chromosome is a small piece of DNA-protein complex existing at the end of linear chromosome in eukaryotic cells. It is about 5-15kb long and consists of a string of "TTAGGG" short fragments repeating in the 5'-3' direction The sequence and the protein that binds to it form a special structure called a telomere to protect the stability of the chromosome and prevent its ends from fusion and degradation. In addition to providing a buffer for non-transcribed DNA, it can also protect the ends of chromosomes from fusion and degeneration. It plays an important role in chromosome positioning, replication, protection and control of cell growth and lifespan, and is associated with apoptosis, cell transformation and immortality. are closely rela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2561/113C12Q2531/113
Inventor 余岚
Owner 浙江指针基因科技有限公司
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