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Kit for co-separation of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from FFPE (formalin fixed and paraffin embedded tissues) as well as method

A paraffin-embedded and formalin technology, applied in the biological field, can solve the problems such as inconsistency and inconsistency of the conclusions, and achieve the effect of improving the yield of RNA

Active Publication Date: 2014-04-16
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because many researchers now want to compare the differences between DNA and RNA for detection or research, but due to the heterogeneity of tumors and other issues, the conclusions are not completely convincing, and there are also discrepancies

Method used

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  • Kit for co-separation of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from FFPE (formalin fixed and paraffin embedded tissues) as well as method
  • Kit for co-separation of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from FFPE (formalin fixed and paraffin embedded tissues) as well as method
  • Kit for co-separation of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from FFPE (formalin fixed and paraffin embedded tissues) as well as method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1: Kit of the present invention

[0129] The kits for co-isolation of DNA and RNA from FFPE samples include:

[0130] ①The lysis buffer RTL, binding buffer Buffer RTB, DNase I working mixture, rinsing buffer RTW and eluent Buffer RTE required for RNA isolation. The Buffer RTL contains 150-300mM Tris·Cl, 0.8-1.2M NaCl, 8-14% (V / V) Tween20, 0.2-0.8% (V / V) sodium dodecyl sulfate; the Buffer RTB contains 50~150mM Tris·Cl, 150~250mM EDTA, 0.8~1.2M KCl, 0.4~1.2M NaCl, 0.1~0.5% (V / V) sodium dodecyl sulfate, 8~ 16% (V / V) Tween20, 4-6M guanidine isothiocyanate; the DNase I working mixture contains DNase I Magic Buffer (80-120mM Tris·Cl, 30-70mM NaCl; 60-100mM MgCl 2 ) and DNase I (3U / μL); the washing solution Buffer RTW contains 250-350 mM Tris·Cl, 30-70 mM EDTA, 0.5-1.5 M NaCl, 70-85% ethanol; the eluent Buffer RTE contains 10-20 mM Tris·Cl, 5-20 mM EDTA.

[0131] Preferably, the Buffer RTL comprises 200 mM Tris·Cl, 1 M NaCl, 10% (V / V) Tween20, 0.5% (V / V) sodium do...

Embodiment 2

[0144] Using the kit described in Example 1, DNA and RNA were isolated from 120 samples and compared with commonly used kits. The samples were paraffin-embedded clinical samples within 3 years, including lung cancer, liver cancer, colorectal cancer, etc.

[0145] 1. Extraction reagents

[0146] (1) RNA isolation

[0147] Lysis buffer RTL: 200mM Tris·Cl, 1M EDTA, 200mM dithiothreitol, 10% (V / V) Tween20, 0.5% (V / V) sodium dodecyl sulfate;

[0148] Binding solution Buffer RTB: 100mM Tris·Cl, 200mM EDTA, 1M KCl, 1M NaCl, 0.2% (V / V) sodium dodecyl sulfate, 10% (V / V) Tween20, 6M Guanidine isothiocyanate;

[0149] DNase I working mixture: DNase I Magic Buffer (100mM Tris Cl, 50mM NaCl, 80mM MgCl 2 ), DNase I (3U / μL);

[0150] Washing solution Buffer RTW: 300 mM Tris Cl, 50 mM EDTA, 1 M NaCl, 10% ethanol;

[0151] Eluent Buffer RTE: 10 mM Tris·Cl, 5 mM EDTA.

[0152] (2) DNA isolation

[0153] Lysis buffer DTL: 100mM Tris Cl, 50mM EDTA, 100mM dithiothreitol, 5% (V / V) sodium dodec...

Embodiment 3

[0196] The kits described in Example 1 and the commonly used kits were thermally accelerated at 56°C for 45 days and used to extract nucleic acids from FFPE samples, and compared with the kits without thermal acceleration.

[0197] 1. Extraction reagents

[0198] (1) RNA isolation

[0199] Lysis buffer RTL: 200mM Tris·Cl, 1M EDTA, 200mM dithiothreitol, 10% (V / V) Tween20, 0.5% (V / V) sodium dodecyl sulfate;

[0200] Binding solution Buffer RTB: 100mM Tris·Cl, 200mM EDTA, 1M KCl, 1M NaCl, 0.2% (V / V) sodium dodecyl sulfate, 10% (V / V) Tween20, 6M Guanidine isothiocyanate;

[0201] DNase I working mixture: DNase I Magic Buffer (100mM Tris Cl, 50mM NaCl, 80mM MgCl 2 ), DNase I (3U / μL);

[0202] Washing solution Buffer RTW: 300 mM Tris Cl, 50 mM EDTA, 1 M NaCl, 10% ethanol;

[0203] Eluent Buffer RTE: 10 mM Tris·Cl, 5 mM EDTA.

[0204] (2) DNA isolation

[0205]Lysis buffer DTL: 100mM Tris Cl, 50mM EDTA, 100mM dithiothreitol, 5% (V / V) sodium dodecyl sulfate, 20% (V / V) Tween20; ...

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Abstract

The invention provides a kit for co-separation of (deoxyribonucleic acid) and RNA (ribonucleic acid) from FFPE (formalin fixed and paraffin embedded tissues) as well as a method. The kit can be used for simultaneously extracting DNA and RNA from an FFPE sample, and has the advantages that not only can nucleic acid extraction be performed simultaneously, time is saved, and the extraction is economical and fast, but also the sample size can be saved, the problem of sample lack in a test can be solved effectively, and further, identical source of the DNA and the RNA used in study can be guaranteed. The kit and the method have the advantages that the operation is simple and direct, the repeatability is high, the yield is high, and enough DNAs and RNAs can be obtained simultaneously only through 2-5 pieces of tumor tissue sections; the obtained nucleic acids are high in purity and has no interference to follow-up detection; and compared with the common kit, the kit has remarkable advantages.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit and method for co-separating DNA and RNA from formalin-fixed paraffin-embedded tissue samples. Background technique [0002] Since Watson and Crick proposed the DNA double helix model in the 1950s, nucleic acid has become the focus of biological research, and has produced the discipline of molecular biology with DNA and RNA as the main research objects. have achieved unprecedented development. In the past 10 years, modern molecular biology techniques have been more and more widely used in various fields of human disease research, accumulating new data for understanding the changes of the genome in pathological conditions. Formalin fixed and paraffin embedded tissues (FFPE, hereinafter referred to as FFPE) are the most commonly used methods for preserving pathological tissues in medical institutions. The large amount of paraffin-embedded tissue accumulated in the archives of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 刘云芬宋庆涛林清华李海燕阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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