Extraction method for total RNA (Ribonucleic Acid) of apple tissues

An extraction method and tissue technology, applied in the direction of recombinant DNA technology, DNA preparation, etc., can solve the problems of high price and achieve the effect of simple and fast operation method, high purity and good integrity

Inactive Publication Date: 2014-04-30
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The extraction effect of commercialized kits is not...

Method used

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  • Extraction method for total RNA (Ribonucleic Acid) of apple tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1) Take 25mg of apple leaves and grind them into powder with liquid nitrogen, add 1000μl of lysate (prepared from the following materials: CTAB: 3%, Tris-HCl (pH8.0): 100mmol / L; EDTA (pH8.0): 25mmol / L L, NaCl: 2mol / L, PVP-40: 2%; β-mercaptoethanol: 4%, dilute to 1000ml) shake and mix immediately.

[0023] 2) Water bath at 65°C for 5 minutes to completely lyse the sample. Centrifuge at 12000rpm at 4°C for 5min, and take the supernatant.

[0024] 3) Add an equal volume of phenol-chloroform mixture (phenol:chloroform=25:24, pH4.5) to the supernatant, mix well, centrifuge at 12000rpm, 4°C for 5min, and take the supernatant.

[0025] 4) Repeat step 3) for the obtained supernatant;

[0026] 5) Add an equal volume of chloroform to the supernatant in step 4), mix well, centrifuge at 12,000 rpm, 4°C for 5 min, and take the supernatant.

[0027] 6) Add 1 / 3 volume of 8mol / L LiCl to the supernatant, mix well, place at -20°C for 1h, centrifuge at 12000rpm at 4°C for 10min, discar...

Embodiment 2

[0035] 1) Take 50mg of apple peel and grind it into powder with liquid nitrogen, add 800μl of lysate (prepared from the following materials: CTAB: 2%, Tris-HCl (pH8.0): 200mmol / L; EDTA (pH8.0): 25mmol / L L, NaCl: 2mol / L, PVP-40: 2%; β-mercaptoethanol: 2%, dilute to 1000ml), shake and mix immediately.

[0036] 2) The sample was completely lysed in a water bath at 65°C for 6 minutes, centrifuged at 12,000 rpm at 4°C for 6 minutes, and the supernatant was taken.

[0037] 3) Add an equal volume of phenol-chloroform (phenol:chloroform = 25:24, pH 4.5) to the supernatant, mix well, centrifuge at 12,000 rpm, 4°C for 5 min, and take the supernatant.

[0038] 4) Repeat step 3) for the obtained supernatant;

[0039] 5) Add an equal volume of chloroform to the supernatant of step 4), mix well, centrifuge at 12,000 rpm, 4°C for 5 min, and take the supernatant.

[0040] 6) Add 1 / 4 volume of 10mol / L LiCl, mix well, place at -20°C for 1h, centrifuge at 12000rpm at 4°C for 10min, discard the...

Embodiment 3

[0048]1) Take 30 mg of apple flower and grind it into powder with liquid nitrogen, add 900 μl of lysate (prepared from the following materials: CTAB: 3%, Tris-HCl (pH8.0): 300 mmol / L; EDTA (pH8.0): 25 mmol / L L, NaCl: 2mol / L, PVP-40: 4%; β-mercaptoethanol: 4%, dilute to 1000ml) shake and mix immediately.

[0049] 2) Water bath at 65°C for 8 minutes to completely lyse the sample. Centrifuge at 12000rpm at 4°C for 5min, and take the supernatant.

[0050] 3) Add an equal volume of phenol-chloroform mixture (phenol:chloroform = 25:24, pH4.5) to the supernatant, mix well, centrifuge at 12000rpm, 4°C for 5min, and take the supernatant.

[0051] 4) Repeat step 3) for the obtained supernatant;

[0052] 5) Add an equal volume of chloroform to the supernatant in step 4), mix well, centrifuge at 12,000 rpm, 4°C for 5 min, and take the supernatant.

[0053] 6) Add 2 / 5 volume of 7mol / L LiCl, mix well, place at -20°C for 1h, centrifuge at 12000rpm, 4°C for 10min, discard the supernatant, ...

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Abstract

The invention discloses an extraction method for total RNA (Ribonucleic Acid) of apple tissues. The method comprises the following steps: grinding the apple tissues by liquid nitrogen to obtain powder; adding lysate and shaking uniformly; completely cracking a sample in a water bath; centrifuging and adding phenol-chloroform with the same volume into supernatant; centrifuging and adding LiCl into the supernatant; centrifuging to obtain an RNA sediment; dissolving the RNA sediment by DEPC (Diethylpyrocarbonate)-treated ddH2O; adding sodium acetate and absolute ethyl alcohol; centrifuging and washing by ethanol to obtain a sediment; and drying the sediment at room temperature to obtain the total RNA of the apple tissues. According to the method, the purity of the extracted RNA is high and the pollution to DNA is not caused; the integrity is good; the operation method is simple, convenient and rapid; the obtained RNA can be used for operation including molecular cloning, Northern blot, Real-time PCR (Polymerase Chain Reaction) and the like.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and in particular relates to a method for extracting apple tissue RNA. Background technique [0002] Apple (Malus domestica) is a plant of the genus Malus in the Rosaceae apple subfamily, a deciduous tree. Apple tissue is rich in polyphenols, polysaccharides, proteins and other secondary metabolites, which seriously affect the extraction of RNA. After being oxidized, phenolic compounds will irreversibly combine with RNA, resulting in the loss of RNA activity; polysaccharides will form insoluble colloids, which will form co-precipitation with RNA; endogenous or exogenous RNases can easily cause RNA degradation; DNA It will also affect the purity of the extracted RNA. Although the guanidinium isothiocyanate (Trizol) method is a recognized method for RNA extraction, it is not suitable for the extraction of RNA from apple tissues because apple tissues are rich in polyphenols and polysaccharides. ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 樊明涛徐颖李亚辉
Owner NORTHWEST A & F UNIV
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