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A method for extracting trace RNA

An extraction method and trace technology, applied in the biological field, can solve the problems of low RNA yield, large initial amount, and many impurities, and achieve the effects of small elution volume, good integrity, and high RNA yield.

Active Publication Date: 2021-02-09
XIAMEN LIFEINT TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention mainly makes improvements to the problems of large initial amount, low RNA yield, many impurities, and inability to extract RNA from trace samples in the traditional RNA extraction method. The RNA isolated from trace samples has high yield and completeness. Well, it meets the needs of downstream experiments

Method used

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  • A method for extracting trace RNA
  • A method for extracting trace RNA
  • A method for extracting trace RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the extraction of trace RNA

[0042] 1. Sample cracking: take 1×10 5 Put a platelet sample (for example: the volume is 3 μL) in a 1.5 mL centrifuge tube, add 4 times the volume of lysate L (12 μL) to the centrifuge tube, vortex to mix, and let stand at room temperature for 5 minutes to completely lyse the platelet and release RNA;

[0043] 2. RNA isolation:

[0044] (2) Add an equal volume (15 μL) of buffer A to the centrifuge tube, invert and mix well, then centrifuge briefly to shake off the liquid;

[0045] (3) Add 1 / 10 volume (3 μL) of magnetic bead B suspension to the centrifuge tube (shake well before use), vortex to mix, and let stand at room temperature for 3 minutes;

[0046] (4) Place the centrifuge tube on the magnetic stand and let it stand for 2 minutes. After the magnetic beads are completely adsorbed on the bottom and wall of the tube, remove the liquid in the centrifuge tube;

[0047] 3. RNA purification:

[0048] (Note: Steps 5-9 can b...

Embodiment 2

[0065] Embodiment 2: Extraction of trace RNA

[0066] 1. Sample cracking:

[0067] (1) Aspirate the medium in the 96-well plate, add 30 μL of lysate L to the 96-well plate to cover the adherent cells in the culture plate (number of cells <100), and let stand at room temperature for 5 minutes to make the cells completely cleaves and releases RNA;

[0068] (2) Mix by blowing and aspirating (try not to generate air bubbles), and carefully transfer all the liquid to a 1.5mL centrifuge tube;

[0069] 2. RNA isolation:

[0070] (3) Add an equal volume (30 μL) of buffer A to the centrifuge tube, invert and mix well, then centrifuge briefly to shake off the liquid;

[0071] (4) Add 1 / 10 volume (6 μL) of magnetic bead B suspension to the centrifuge tube (shake well before use), vortex to mix, and let stand at room temperature for 3 minutes;

[0072] (5) Place the centrifuge tube on the magnetic stand and let it stand for 2 minutes. After the magnetic beads are completely adsorbed o...

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Abstract

The invention discloses a method for extracting trace RNA. Steps: sample lysis: completely lyse platelets and release RNA; RNA isolation: use nucleic acid-assisted precipitant combined with magnetic beads to separate RNA from the sample; wherein nucleic acid-assisted precipitant is glycogen; RNA purification: remove non-specific binding to DNA and protein impurities from magnetic beads. The present invention provides a micro-RNA extraction method suitable for platelets, aiming at extracting as low as 1×10 5 High-quality and sufficient RNA was isolated from platelets or 10 cells for downstream experiments and analysis.

Description

technical field [0001] The invention relates to the biological field, in particular to a method for extracting trace RNA. Background technique [0002] RNA extraction is the basis of molecular biology research focusing on RNA research, and is a key step in the study of gene expression. Platelet RNA detection, as a new liquid biopsy technology, has extremely important application prospects in cancer screening and cardiovascular disease research and monitoring. 1×10 5 The total RNA content in a platelet is about 100pg, and its quality is only equivalent to the RNA of 10 white blood cells. How to extract high-quality RNA from very small initial samples for downstream experiments is an urgent problem to be solved. [0003] Traditional RNA extraction methods such as phenol-chloroform method and CTAB method require a large initial sample volume, which needs to be greater than 1×10 5 A cell or 5mg of tissue cannot be extracted from a small amount of sample RNA. The existing ad...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 陈欣欣叶国栋许剑雄陈茂立韩大雄郭奇伟肖剑萍蔡逸民杨燕燕李顺杰董康梅朱莎莎张丽芳宋丹
Owner XIAMEN LIFEINT TECH CO LTD
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