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Method for separating nucleic acids from fepe tissue

A tissue and nucleic acid technology, applied in the field of nucleic acid isolation to achieve high yield

Active Publication Date: 2018-04-17
THE ASAN FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the nucleic acids in FFPE samples are broken down into small fragments and entangled with proteins, there are limitations in isolating DNA from FFPE tissues at the molecular level for clinical applications (Feldman MY et al., Prog Nucleic Acid Res Mol Biol, (1973) 13,1-49)

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  • Method for separating nucleic acids from fepe tissue
  • Method for separating nucleic acids from fepe tissue
  • Method for separating nucleic acids from fepe tissue

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Example 1: Isolation of Genomic DNA (Including Removal of Paraffin) from FFPE Tissue Samples

[0070] (1) FFPE block fragments and paraffin removal

[0071] Cut the FFPE block to a thickness of 6 μm, then place one or two FFPE tissue segments in a 1.5 mL tube. Add 1 mL of xylene and mix, then centrifuge at 12,000 x g for 3 min at room temperature. After removing the supernatant, 0.8 mL of xylene and 0.4 mL of EtOH were added and mixed, centrifuged at 12000×g for 3 minutes at room temperature, and the supernatant was removed. Add 1 mL of EtOH and mix again, centrifuge at 12,000 x g for 3 min at room temperature, and remove the supernatant. In order to completely evaporate the remaining EtOH, incubation was performed at room temperature for 0.5 h to 1 h.

[0072] (2) Cleavage and Genomic DNA Isolation

[0073] Add 90 μL of lysis buffer (10 mM Tris-Cl, pH 8.0, 100 mM EDTA, pH 8.0, 0.5% SDS) and 10 μL of proteinase K to the tube containing the paraffin-depleted FFPE tis...

Embodiment 2

[0077] Example 2: Isolation of Genomic DNA from FFPE Tissue Samples (without Paraffin Removal)

[0078] (1) FFPE block fragmentation

[0079] Cut the FFPE block to a thickness of 6 μm, then place one or two FFPE tissue segments in a 1.5 mL tube.

[0080] (2) Cleavage and Genomic DNA Isolation

[0081] Add 200 μL of lysis buffer (10 mM Tris-Cl, pH 8.0, 100 mM EDTA, pH 8.0, 0.5% SDS), 200 μL of mineral oil, and 10 μL of proteinase K to the tube containing the FFPE tissue fragment and mix, then incubate at 56 °C Incubate overnight.

[0082] Next, the lower phase was separated and transferred to a new tube. 100 μL magnetic beads (AMPure XP) and 100 μL solution A (2.5M NaCl, 20% PEG-8000) were added to the tube and mixed. Tubes were then incubated at room temperature for 3 minutes. Tubes were incubated on a magnetic stand for 5 minutes and the supernatant removed.

[0083] After adding 500 μL of washing buffer (85% EtOH) and reacting, the ethanol supernatant was removed on a ...

Embodiment 3

[0086] Embodiment 3: Quantitative analysis of DNA

[0087] (1) Quantification using NanoDrop (ND)

[0088] NanoDrop 2000 (Thermo Scientific) was used in the dsDNA concentration measurement mode. After measuring the blank value using the DNA extraction eluate as a reference, use 1 μL of the extracted DNA to measure the OD 260 Under the absorbance, the concentration was calculated using the following formula: dsDNA concentration = absorbance (OD 260 )*50ng / μL. Calculate the final yield by multiplying by 100 μL (total volume of extraction solvent).

[0089] (2) Quantification using PicoGreen (PG)

[0090] Quant-Ti PicoGreen dsDNA assay kit (Invitrogen) was purchased for measuring dsDNA concentration.

[0091] A, the preparation of PicoGreen reagent

[0092] Thirty minutes before measuring the concentration, dilute the PicoGreen reagent to 1 / 200 with 1×TE buffer, wrap it in aluminum foil and store it in the dark at room temperature.

[0093] B. Preparation of standard conce...

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Abstract

The present invention relates to: a method for separating nucleic acids from a sample containing formalin-fixed, paraffin-embedded (FFPE) tissue sections; a nucleic acid separation kit; and a lysis buffer for separating nucleic acids.

Description

technical field [0001] The present invention relates to a method for isolating nucleic acid from a sample prepared by lysing formalin-fixed paraffin-embedded (FFPE) tissue fragments, a kit for isolating nucleic acid, and a lysis buffer for isolating nucleic acid. Background technique [0002] Recently, molecular biology techniques have been widely used in the medical field. Especially, in terms of molecular pathology, these technologies can perform pathological diagnosis from the morphological level to the gene level, and can also reveal the genetic characteristics of specific diseases at the morphological level and the gene level (DeMarzo et al., Lancet, (2003) 361, 955-64). For such a diagnostic procedure, the hospital collects tissue in the form of formalin-fixed paraffin-embedded (FFPE), which enables analysis and diagnosis of genetic information associated with various clinical symptoms (Roukos DH et al., Pharmacogenomics, (2010) 11, 283-7 ). However, since nucleic a...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806C12N15/1013C12N15/10
Inventor 张世镇千成民金兑任李政慎崔恩卿
Owner THE ASAN FOUND
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