mRNA (messenger ribonucleic acid) quick-extraction kit

Abstract

The invention relates to an mRNA (messenger ribonucleic acid) quick-extraction kit and a preparation method thereof. The kit comprises a separation device for solid-liquid separation, wherein the separation device at least comprises a layer of cellulose solid-phase carrier. The kit also comprises at least one tube containing a cell or tissue lysis solution, at least one tube containing a biotinylated Oligo (dT) probe, at least one tube containing a combination buffer solution, and at least one tube containing an elution buffer solution. The kit is simple, quick and efficient to operate, and is suitable for separating and extracting mRNA in a biological sample. The invention is mainly characterized in that the separation device uses cellulose as the solid-phase carrier, a fusion protein CBD-SA is used as a biological bridging agent, and a biologically-specific coupling method is used instead of the traditional physical adsorption method and chemical bonding method to activate the cellulose solid-phase carrier, so that the activated solid-phase carrier can be efficiently combined with the biotinylated reagent to be used for separating protein or nucleic acid.

Description

technical field

[0001] The invention relates to a rapid mRNA extraction kit and a preparation method thereof, belonging to the field of biotechnology. Background technique

[0002] mRNA is short for messenger RNA, or messenger RNA. mRNA is transcribed from DNA, carrying the corresponding genetic information, and providing the necessary information for the next step of translation into protein. At present, with the increasing development of molecular biology, efficient extraction of complete mRNA from eukaryotic cells or tissues is the key to functional genomics applications such as cDNA library construction, EST sequencing, Northern dot hybridization, microarray gene expression analysis, and quantitative PCR. step. In mammals, a large number of RNA molecules exist in the form of tRNA and ribosomal RNA, while mRNA only accounts for 1-5% of the total RNA.

[0003] Traditional mRNA extraction includes two steps; first, total RNA is extracted from cells or tissues using metho...

Examples

Embodiment 1

[0039] Example 1. Activation and assembly of cellulose filter paper (i.e. preparation of bioactivated cellulose solid phase carrier)

[0040] First prepare the fusion protein CBD-SA (see Chinese patent CN101640085B).

[0041] Then, at 2-8°C, take a certain amount of cellulose filter paper, add the fermented lysate of the recombinant fusion protein CBD-SA, oscillate for sufficient reaction, rinse with PBS buffer repeatedly for 8 times, and then dry at room temperature Dry it. by figure 2 The double-layer microcentrifuge tube of B is taken as an example. The bioactivated cellulose filter paper is cut into a circle with a suitable diameter for the inner tube, and placed flat on the bottom of the inner separation tube and on the upper end of the microporous membrane or screen-like structure. , with a thickness of 1 mm, about 5 layers of filter paper are placed.

Embodiment 2

[0042] Activation and assembly of embodiment 2 cellulose absorbent cotton

[0043] Referring to Example 1, at 2-8°C, take a certain amount of absorbent cotton, add the fermented cell lysate of the recombinant fusion protein CBD-SA, oscillate for sufficient reaction, rinse with PBS buffer repeatedly for 8 times, and then dry at room temperature , and pressed into flakes. Cut the above-mentioned bioactivated absorbent cotton into a circle suitable for the diameter of the inner tube, lay it flat and tightly fix the bottom of the inner separation tube, and the upper end of the microporous membrane or screen-like structure, with a thickness of about 2mm.

Embodiment 3

[0044] The extraction of mRNA in the tissue sample of embodiment 3

[0045] The rat was anesthetized and dissected, and 50 mg of heart tissue was taken and chopped, placed in a lysate (4M guanidine isothiocyanate, 25 mM sodium citrate and 1% β-mercaptoethanol, pH7.0), homogenized and pulverized, and then added 1mL binding buffer (75mM NaCl, 8.5mM sodium citrate, pH 7.2), mix by inversion, then add 60pM biotinylated Oligo(dT) probe, mix well, and incubate at 70°C for 5min. Then at room temperature, 12000rpm, centrifuge for 10min, carefully collect the supernatant, transfer to the inner layer separation tube of Example 1 activated with cellulose filter paper, and centrifuge at room temperature for 1min at 12000rpm. Discard the waste liquid in the outer tube; add 600 μL of SSC buffer solution to the inner tube, centrifuge at 12000 rpm at room temperature for 30-60 s, discard the waste liquid in the collection tube, and repeat this step twice. Finally, put the inner tube into a n...

technical field

[0001] The invention relates to a rapid mRNA extraction kit and a preparation method thereof, belonging to the field of biotechnology. Background technique

[0002] mRNA is short for messenger RNA, or messenger RNA. mRNA is transcribed from DNA, carrying the corresponding genetic information, and providing the necessary information for the next step of translation into protein. At present, with the increasing development of molecular biology, efficient extraction of complete mRNA from eukaryotic cells or tissues is the key to functional genomics applications such as cDNA library construction, EST sequencing, Northern dot hybridization, microarray gene expression analysis, and quantitative PCR. step. In mammals, a large number of RNA molecules exist in the form of tRNA and ribosomal RNA, while mRNA only accounts for 1-5% of the total RNA.

[0003] Traditional mRNA extraction includes two steps; first, total RNA is extracted from cells or tissues using metho...

Examples

Embodiment 1

[0039] Example 1. Activation and assembly of cellulose filter paper (i.e. preparation of bioactivated cellulose solid phase carrier)

[0040] First prepare the fusion protein CBD-SA (see Chinese patent CN101640085B).

[0041] Then, at 2-8°C, take a certain amount of cellulose filter paper, add the fermented lysate of the recombinant fusion protein CBD-SA, oscillate for sufficient reaction, rinse with PBS buffer repeatedly for 8 times, and then dry at room temperature Dry it. by figure 2 The double-layer microcentrifuge tube of B is taken as an example. The bioactivated cellulose filter paper is cut into a circle with a suitable diameter for the inner tube, and placed flat on the bottom of the inner separation tube and on the upper end of the microporous membrane or screen-like structure. , with a thickness of 1 mm, about 5 layers of filter paper are placed.

Embodiment 2

[0042] Activation and assembly of embodiment 2 cellulose absorbent cotton

[0043] Referring to Example 1, at 2-8°C, take a certain amount of absorbent cotton, add the fermented cell lysate of the recombinant fusion protein CBD-SA, oscillate for sufficient reaction, rinse with PBS buffer repeatedly for 8 times, and then dry at room temperature , and pressed into flakes. Cut the above-mentioned bioactivated absorbent cotton into a circle suitable for the diameter of the inner tube, lay it flat and tightly fix the bottom of the inner separation tube, and the upper end of the microporous membrane or screen-like structure, with a thickness of about 2mm.

Embodiment 3

[0044] The extraction of mRNA in the tissue sample of embodiment 3

[0045] The rat was anesthetized and dissected, and 50 mg of heart tissue was taken and chopped, placed in a lysate (4M guanidine isothiocyanate, 25 mM sodium citrate and 1% β-mercaptoethanol, pH7.0), homogenized and pulverized, and then added 1mL binding buffer (75mM NaCl, 8.5mM sodium citrate, pH 7.2), mix by inversion, then add 60pM biotinylated Oligo(dT) probe, mix well, and incubate at 70°C for 5min. Then at room temperature, 12000rpm, centrifuge for 10min, carefully collect the supernatant, transfer to the inner layer separation tube of Example 1 activated with cellulose filter paper, and centrifuge at room temperature for 1min at 12000rpm. Discard the waste liquid in the outer tube; add 600 μL of SSC buffer solution to the inner tube, centrifuge at 12000 rpm at room temperature for 30-60 s, discard the waste liquid in the collection tube, and repeat this step twice. Finally, put the inner tube into a n...

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