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Method for quickly separating and detecting crosslinking DNA in paraffin-embedded tissue sample

A technology for tissue samples and paraffin embedding, which is applied in the direction of recombinant DNA technology, DNA preparation, microbial determination/inspection, etc., and can solve the problems of low recovery efficiency, time-consuming, unfavorable large sample screening, etc.

Inactive Publication Date: 2016-07-20
BEIJING JIAOTONG UNIV +1
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Problems solved by technology

At present, there is basically no effective monitoring method for the storage of paraffin-embedded tissue samples, because the DNA isolation kits for paraffin-embedded tissue samples on the market are either time-consuming, or the recovery efficiency is low, and most importantly, they are expensive, which is not conducive to large samples. screening, testing or monitoring

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  • Method for quickly separating and detecting crosslinking DNA in paraffin-embedded tissue sample

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Embodiment 1

[0030] Operation method:

[0031] 1) Take 1 / 8~1 piece of paraffin-embedded sample and add ddH 2 O, cover, 70°C, 5-10min, discard the supernatant;

[0032] 2) Add 1-5μL 5mg / mL proteinase K, add ddH 2 0 to 10 μL, 55°C, 30min;

[0033] 3) Add 10 μL of 10% Chelex100 microbeads, shake vigorously for 10 seconds, and centrifuge at 300×g;

[0034] 4) High temperature vibration incubation, the conditions are: 95°C, 1300 rpm, 10min;

[0035] 5) Centrifugation, the centrifugal force is 3000-14000×g, and the room temperature is 10sec;

[0036] 6) Transfer 20 μL of supernatant to a new centrifuge tube, add 2 μL of RNAseA, and 4 μL of 6× Loading Buffer, and mix;

[0037] 7) Gel analysis with 1.2% gel.

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Abstract

A method for quickly separating and detecting crosslinking DNA in a paraffin-embedded tissue sample comprises the following steps: adding ddH2O into the paraffin-embedded tissue sample, incubating at 60-80 DEG C, and removing supernate; adding a protein degrading agent, and incubating; adding a DNA de-crosslinking and separating and extracting agent, and vibrating, the DNA de-crosslinking and separating and extracting agent being Chelex 100; vibrating and incubating at a high temperature; centrifuging and reserving supernate; adding a sampling bearing buffer into the supernate; detecting electrophoretically. In the method, it is possible to estimate degradation degree and completeness of genome DNA of the tissue sample and determine the quality and uses of the sample through fragmented crosslinking DNA of the paraffin-embedded sample subjected to quick quality control; the method of the invention is a quick, low-rice and effective quality control detection method for paraffin-embedded sample DNA.

Description

technical field [0001] The invention relates to the field of rapid separation and detection of genomic DNA, and more specifically, to the quality control and evaluation of DNA in formalin-fixed and paraffin-embedded tissue samples. Background technique [0002] Formalin-fixed and paraffin-embedded tissues are the most common processing and storage methods for clinical pathological tissue specimens. A large number of paraffin-embedded tissue specimens are produced continuously from various clinical hospitals every day. DNA extraction from paraffin-embedded tissue samples is its most widespread and primary use. However, with the passage of time, coupled with factors such as the quality of the specimen before processing and the processing method itself, many specimen tissues will be degraded to varying degrees, including the degradation of DNA, that is, "fragmentation". On the one hand, fragmented DNA causes tissue cells to lose the integrity of their genetic DNA, which will ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
CPCC12Q1/6806
Inventor 黄家强景坤玉林舒晔林博楠
Owner BEIJING JIAOTONG UNIV
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