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Method for complete cryopreservation and thawing of intracellular RNC (ribosome nascent-chain complex)

A cryopreservation and ribosome technology, applied in the field of life sciences, can solve problems such as difficulties in commercial services, inability to store and transport for a long time, and inability to achieve effective preservation

Active Publication Date: 2014-12-10
武汉承启医学科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is only suitable for extracting RNC from fresh cells
Because RNC is very fragile, it cannot be stored and transported without freezing for a long time; after freezing and thawing, ribosomes will fall off a large number of mRNAs, which cannot be effectively preserved (Zhang et al., 2009)
Therefore, RNC-related analysis can only be done in a small number of laboratories with all relevant experimental conditions, and cannot be preserved and transported
This poses great difficulties for many experimental applications as well as commercial services

Method used

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  • Method for complete cryopreservation and thawing of intracellular RNC (ribosome nascent-chain complex)
  • Method for complete cryopreservation and thawing of intracellular RNC (ribosome nascent-chain complex)

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Embodiment 1

[0046] (1) Use cell culture medium at 37°C, 0.5% CO 2 The human lung cancer cell line A549 was cultured in the environment.

[0047] (2) Then add cycloheximide aqueous stock solution, shake well, the final concentration of cycloheximide is 0.1mg / ml.

[0048] (3) In the original culture environment (i.e. 37°C, 0.5% CO 2 environment) and continue to incubate for 15 minutes.

[0049] (4) Pour out the culture medium carefully, and then add the PBS solution pre-cooled to 0°C, the function of the PBS solution is to wash the cells.

[0050] (5) Pour out the PBS solution carefully, and then add the PBS solution pre-cooled to 0°C. At this time, the PBS solution must submerge the A549 cells.

[0051] (6) Since A549 cells grow adherently, put the cell adherent surface of the cell culture flask into liquid nitrogen quickly until the temperature stabilizes at the liquid nitrogen temperature (-196°C).

[0052] (7) Quickly transfer the quick-frozen cell culture bottle to a -80°C ultra-lo...

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Abstract

The invention discloses a method for complete cryopreservation and thawing of an RNC (intracellular ribosome nascent-chain complex). The method comprises the following steps: adding a translation elongation inhibitor into cell culture fluid in normal growth, cooling cells to the temperature of 0-4 DEG C after the translation elongation inhibitor effectively acts on the RNC and translation elongation is inhibited, cleaning the cells by using a PBS solution which is precooled to the temperature of 0-4 DEG C, adding the PBS solution into the cells, rapidly freezing in liquid nitrogen, preserving at an ultra-low temperature, thawing on ice, and extracting the RNC. According to the method, ribosome is effectively fixed by adopting the translation elongation inhibitor and is unlikely to dissociate, and the translation conditions in the cells are fixed. Moreover, the RNC is effectively cryopreserved for long-term storage and transport through a liquid nitrogen quick freezing method, and the extracted RNC after thawing is the same as the RNC extracted from fresh cells, so the limitation of time and space is broken through, and convenience is provided for research of the RNC and development of commercial service of the RNC.

Description

technical field [0001] The invention belongs to the technical field of life sciences, and in particular relates to a method for intact cryopreservation and thawing of ribosome nascent-chain complexes (Ribosome Nascent-chain Complex, RNC). Background technique [0002] Translation regulation is an important regulatory level in life. In cells, some genes are translated more, some genes are translated less, and some genes are not translated at all. Therefore, it is extremely necessary to study the translation status in cells. When the cell is translating, the ribosome binds to the mRNA chain and moves, and gradually synthesizes the protein polypeptide chain (called nascent-chain) according to the triplet codon information on the mRNA. This complex is called ribosome nascent-chain complex (Ribosome Nascent-chain Complex, RNC). Only when the RNC is completely extracted can the qualitative and quantitative research on the mRNA being translated and the research on the nascent pe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 孙黎张弓王通金静洁
Owner 武汉承启医学科技有限公司
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