CN112710523B - Collecting device suitable for molecular marker detects - Google Patents
Collecting device suitable for molecular marker detects Download PDFInfo
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- CN112710523B CN112710523B CN202011606511.XA CN202011606511A CN112710523B CN 112710523 B CN112710523 B CN 112710523B CN 202011606511 A CN202011606511 A CN 202011606511A CN 112710523 B CN112710523 B CN 112710523B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The application belongs to the technical field of plant identification, and discloses a collecting device suitable for detecting molecular markers, wherein a sample extracting module is used for collecting plant DNA samples, a nuclear cell sample collecting module is used for collecting nuclear cells, a nuclear cell sample cleaning and storing module is used for cleaning and freezing nuclear cells and other impurities, a sample grinding and processing module and a sample homogenizing and processing module are controlled to conduct grinding and homogenizing treatment on samples, and an RNA eluting module and an RNA marker detecting module are used for eluting separated RNA and measuring the concentration of the RNA. The application converts the obtained DNA fingerprint information into the unique molecular identity card of each material by utilizing the bar code technology, so that the molecular difference between different plants is visible and is rapidly identified by a machine, the influence of factors such as environment, human factors and the like is eliminated, the accuracy of a detection result is ensured, the detection efficiency is improved, and the detection sensitivity is high.
Description
Technical Field
The application belongs to the technical field of plant identification, and particularly relates to a collecting device suitable for molecular marker detection.
Background
At present, the rapid development of molecular biology greatly promotes the application of DNA barcoding (DNA barcoding) in the field of plant identification. The DNA bar code identification is quick and accurate, is hopeful to realize automatic identification, and is an effective supplement of the traditional plant identification method. The technology is a novel molecular diagnosis technology for rapidly and accurately identifying species by using one or more standard DNA sequences as markers. ITS2 sequences are widely focused by researchers at home and abroad by virtue of the characteristics of shorter sequence fragments, quicker species level variation and the like. The combined use of ITS2 and gene probes will allow for faster and more convenient species identification. The combined use of ITS2 and probes allows for faster and more convenient identification of these two primitive species.
The establishment of the DNA molecular marker technology is a major breakthrough of a research method of plant resource science, plays an important role in plant germplasm resource identification, and especially opens up a new path for classification and identification of medicinal plant germplasm resources. The identification of the germplasm resources of the medicinal plants is the basis of the research of the medicinal plants, and has important significance for determining the original plants, ensuring the drug effect, reasonably preserving and utilizing the existing germplasm resources and the like. The molecular biology can expand biological characters from characterization to biological information macromolecules, and can control the quality of traditional Chinese medicine at molecular level by utilizing DNA, RNA and protein to reflect the transmission sequence of genetic codes, and is also the most valuable classification and identification index of the Chinese herbal medicine genuine property.
When detecting molecular markers, the collection operation needs to be performed firstly, but the existing molecular marker detection device lacks a collection function, and often needs to increase operations such as transferring in the operation process, so that the detection efficiency is reduced. In addition, the existing detection device of the molecular marker has low sensitivity, and the detection cannot be performed when the content of the molecular marker is too small, so that the accuracy of plant identification is affected.
Through the above analysis, the problems and defects existing in the prior art are as follows:
(1) The existing molecular marker detection device lacks a collection function, so that operations such as transferring and the like are required to be added in the operation process, and the detection efficiency is reduced;
(2) The existing detection device for the molecular marker has low sensitivity, and the detection cannot be performed when the content of the molecular marker is too small, so that the accuracy of plant identification is affected.
Disclosure of Invention
Aiming at the problems existing in the prior art, the application provides a collecting device suitable for molecular marker detection.
The application is realized in that a collecting device suitable for molecular marker detection specifically comprises:
the sample extraction module is connected with the central control module and comprises a sampling needle and a sampling test tube, and is used for collecting plant DNA samples prepared in advance through sampling, and the collected plant DNA samples and an anticoagulant are simultaneously input into the sampling test tube on the oscillator;
the nuclear cell sample collection module is connected with the central control module and comprises a centrifuge and a straw, the centrifugal processing is carried out on the uncoagulated plant DNA sample through the centrifuge, the centrifuged nuclear cell mixed solution is sucked out through the straw, and the nuclear cell mixed solution is transferred into a sampling test tube with a culture solution for storage;
the nuclear cell sample cleaning and storing module is connected with the central control module and comprises a centrifugal machine, the centrifugal machine is used for carrying out centrifugal treatment on the nuclear cell mixed solution in the sampling test tube, the supernatant is removed, and the steps are repeated for five times in sequence to obtain pure nuclear cells;
the sample grinding treatment module is connected with the central control module and comprises a grinder, frozen nuclear cell sample blocks are directly placed into a mortar of the grinder, liquid nitrogen is added into the mortar, and the mortar is quickly ground;
the sample homogenizing treatment module is connected with the central control module and comprises a homogenizer, a uniformly ground sample is placed in a centrifuge tube of the homogenizer, an RNA extraction reagent is added into the centrifuge tube, the centrifuge tube is subjected to vacuum treatment, and the mixed solution in the centrifuge tube is subjected to homogenizing treatment through the homogenizer;
the separation and extraction module is connected with the central control module and comprises a centrifugal machine, and is used for separating and extracting RNA from the homogenized mixed solution;
the RNA eluting module is connected with the central control module and comprises an oscillator and a centrifugal machine, and is used for eluting the separated RNA through the oscillator;
the RNA marker detection module is connected with the central control module and comprises a spectrophotometer, and the spectrophotometer is used for measuring the concentration of RNA;
the central control module is connected with the sample extraction module, the nuclear cell sample collection module, the nuclear cell sample cleaning and storage module, the sample grinding and processing module, the sample homogenizing and processing module, the separation and extraction module, the RNA eluting module, the RNA marker detection module and the power supply module and is used for controlling the sample extraction module to collect and anticoagulate a plant DNA sample, the nuclear cell sample collection module to collect nuclear cells, the nuclear cell sample cleaning and storage module to clean the nuclear cells and other impurities and store the nuclear cells in a freezing way, the sample grinding and processing module and the sample homogenizing and processing module to grind the samples in a homogenizing way, and the RNA eluting module and the RNA marker detection module to elute the separated RNA and measure the concentration of the RNA;
the power supply module is connected with the sample extraction module, the nuclear cell sample collection module, the nuclear cell sample cleaning and storing module, the sample grinding and processing module, the sample homogenizing and processing module, the separation and extraction module, the RNA eluting module, the RNA marker detection module and the central control module and is used for providing power support for the normal operation of instruments or equipment in each module.
Further, the rotational speed of the centrifugation treatment of the plant DNA sample in the nuclear cell sample collection module is 1500rpm for 25min.
Further, the rotational speed of the nuclear cell sample cleaning and storing module for carrying out centrifugal treatment on the nuclear cell mixed solution is 1200rpm, and the centrifugal time is 20min.
Further, the homogenizing temperature in the sample homogenizing treatment module is 10-25 ℃ and the time is 5-10min.
The application also aims at providing a collecting method suitable for molecular marker detection, which comprises the following specific steps:
firstly, collecting plant DNA samples by sampling, simultaneously inputting the collected plant DNA samples and an anticoagulant into a sampling test tube arranged on an oscillator, and fully mixing the plant DNA samples and the anticoagulant under the action of oscillation of the oscillator;
centrifuging the uncondensed plant DNA sample by a centrifuge, sucking out the centrifuged nuclear cell mixed solution by a suction pipe, and transferring the nuclear cell mixed solution into a sampling test tube with a culture solution for storage;
step three, centrifuging the nuclear cell mixed solution in the sampling test tube by using a centrifuge, removing supernatant, and repeating the steps for five times in sequence to obtain pure nuclear cells;
step four, directly placing the frozen nuclear cell sample block into a mortar of a grinder, adding liquid nitrogen, and quickly grinding;
placing the uniformly ground sample into a centrifuge tube of a homogenizer, adding an RNA extraction reagent into the centrifuge tube, carrying out vacuum treatment on the centrifuge tube, and carrying out homogenization treatment on the mixed solution in the centrifuge tube through the homogenizer;
step six, separating and extracting RNA from the homogenized mixed solution; eluting the separated RNA by an oscillator; the determination of the RNA concentration was accomplished using a spectrophotometer.
Further, in the first step, the preparation method of the plant DNA sample comprises:
s1, taking 5.0 g of young leaves of plants, putting the young leaves into liquid nitrogen, freezing and grinding the young leaves into powder;
s2, transferring the mixture into 12ml of an extracting solution preheated at 65 ℃ for 20 minutes, and then adding chloroform with the same volume for uniform mixing;
s3, oscillating for 15 minutes at normal temperature and low speed, centrifuging for 15 minutes at 12000 r/min, taking supernatant, and adding 2/3 volume of isopropanol to precipitate DNA;
s4, taking out flocculent DNA, washing the flocculent DNA twice with 70% ethanol, naturally drying the flocculent DNA, and adding 500 μl of buffer solution with pH of 8.0TE for dissolution and quantification for later use.
In the sixth step, the separation and extraction of RNA specifically includes:
firstly, filling the homogenized mixed solution into a centrifuge tube, adding chloroform into the centrifuge tube, fully oscillating and uniformly mixing in an oscillator, and then standing for 15min at room temperature;
secondly, centrifuging for 15min under the action of a centrifugal force of 12000g at the temperature of 4 ℃, sucking supernatant fluid after the centrifugation is finished, transferring the supernatant fluid into a new centrifuge tube, adding isopropanol, uniformly mixing, and standing at room temperature;
thirdly, centrifuging for 15min under the action of 12000g of centrifugal force at 4 ℃, and discarding supernatant after the centrifugation is finished to obtain the RNA after precipitation.
Further, in the second step, the standing time at room temperature is 10-15min.
In step six, the elution of RNA specifically includes:
(1) Adding 75% ethanol into the precipitated RNA centrifuge tube, and oscillating the centrifuge tube with low power to suspend and precipitate RNA in the centrifuge tube;
(2) Centrifuging the suspension precipitate in a centrifuge tube at 4deg.C under the action of 8000g centrifugal force for 15-20min, removing supernatant, and air drying at room temperature.
Further, in the sixth step, the method for measuring the concentration of RNA by using a spectrophotometer includes:
1) Adding 0.5-2 mu l of ultrapure water into a detection eye by using a pipetting gun, resetting a signal receiver, and operating an "Ok" key of operation software to enable the instrument to enter an initialized state;
2) Loading a control sample, and running an operation software 'Blank' key;
3) And loading the eluted RNA, and running an operation software 'Measure' key, wherein the concentration of the RNA is instantly displayed on a display screen.
By combining all the technical schemes, the application has the advantages and positive effects that:
the application converts the obtained DNA fingerprint information into the unique molecular identity card of each material by utilizing the bar code technology, so that the molecular difference between different plants is visible and is rapidly identified by a machine, the influence of factors such as environment, human factors and the like is eliminated, and the accuracy of a detection result is ensured.
The application integrates the sample collection, processing and detection functions in the molecular marker detection process, improves the detection efficiency, and has high detection sensitivity and high detection result precision.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a block diagram of a collection device suitable for molecular marker detection according to an embodiment of the present application;
in the figure: 1. a sample extraction module; 2. a nuclear cell sample collection module; 3. a nuclear cell sample cleaning storage module; 4. a sample grinding treatment module; 5. a sample homogenizing treatment module; 6. a separation and extraction module; 7. an RNA elution module; 8. an RNA marker detection module; 9. a central control module; 10. and a power supply module.
FIG. 2 is a flow chart of a collection method suitable for molecular marker detection according to an embodiment of the present application.
Fig. 3 is a flowchart of a method for separating and extracting RNA according to an embodiment of the present application.
FIG. 4 is a flow chart of an elution method for RNA according to an embodiment of the present application.
FIG. 5 is a flow chart of a method for RNA concentration detection using a spectrophotometer according to an embodiment of the present application.
FIG. 6 is a flowchart of a method for preparing a plant DNA sample according to an embodiment of the present application.
Detailed Description
The present application will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
In view of the problems of the prior art, the present application provides a collecting device suitable for detecting molecular markers, and the present application is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, a collecting device for detecting a molecular marker according to an embodiment of the present application includes:
the sample extraction module 1 is connected with the central control module and comprises a sampling needle and a sampling test tube, and is used for collecting plant DNA samples prepared in advance through sampling, and the collected plant DNA samples and an anticoagulant are simultaneously input into the sampling test tube on the oscillator;
the nuclear cell sample collection module 2 is connected with the central control module and comprises a centrifuge and a straw, the centrifugal treatment is carried out on the uncoagulated plant DNA sample through the centrifuge, the centrifugal nuclear cell mixed liquid is sucked out through the straw, and the nuclear cell mixed liquid is transferred into a sampling test tube with a culture solution for storage;
the nuclear cell sample cleaning and storing module 3 is connected with the central control module and comprises a centrifugal machine, the centrifugal machine is used for carrying out centrifugal treatment on the nuclear cell mixed liquid in the sampling test tube, the supernatant is removed, and the steps are repeated for five times in sequence to obtain pure nuclear cells;
the sample grinding treatment module 4 is connected with the central control module and comprises a grinder, frozen nuclear cell sample blocks are directly put into a mortar of the grinder, liquid nitrogen is added into the mortar, and the mortar is quickly ground;
the sample homogenizing treatment module 5 is connected with the central control module and comprises a homogenizer, a uniformly ground sample is placed in a centrifuge tube of the homogenizer, an RNA extraction reagent is added into the centrifuge tube, the centrifuge tube is subjected to vacuum treatment, and the mixed solution in the centrifuge tube is subjected to homogenizing treatment through the homogenizer;
the separation and extraction module 6 is connected with the central control module and comprises a centrifugal machine for separating and extracting RNA from the homogenized mixed solution;
the RNA eluting module 7 is connected with the central control module and comprises an oscillator and a centrifugal machine, and is used for eluting the separated RNA through the oscillator;
the RNA marker detection module 8 is connected with the central control module and comprises a spectrophotometer, and the spectrophotometry is used for measuring the concentration of RNA;
the central control module 9 is connected with the sample extraction module, the nuclear cell sample collection module, the nuclear cell sample cleaning and storing module, the sample grinding and processing module, the sample homogenizing and processing module, the separation and extraction module, the RNA eluting module, the RNA marker detection module and the power supply module and is used for controlling the sample extraction module to collect and anticoagulate a plant DNA sample, the nuclear cell sample collection module to collect nuclear cells, the nuclear cell sample cleaning and storing module to clean the nuclear cells and other impurities and store the nuclear cells in a freezing way, the sample grinding and processing module and the sample homogenizing and processing module to grind and homogenize the samples, and the RNA eluting module and the RNA marker detection module to elute the separated RNA and measure the concentration of the RNA;
the power supply module 10 is connected with the sample extraction module 1, the nuclear cell sample collection module 2, the nuclear cell sample cleaning and storage module 3, the sample grinding and processing module 4, the sample homogenizing and processing module 5, the separation and extraction module 6, the RNA elution module 7, the RNA marker detection module 8 and the central control module 9 and is used for providing power support for the normal operation of instruments or equipment in each module.
The rotational speed of the centrifugation treatment of peripheral venous blood in the nuclear cell sample collection module 2 in the embodiment of the application is 1500rpm for 25min.
The rotational speed of the nuclear cell sample cleaning and storing module 3 for carrying out centrifugal treatment on the nuclear cell mixed liquid is 1200rpm, and the centrifugal time is 20min.
The homogenization temperature in the sample homogenization treatment module 5 in the embodiment of the application is 10-25 ℃ and the time is 5-10min.
As shown in fig. 2, the method for collecting the molecular marker provided by the embodiment of the application specifically comprises the following steps:
s101, collecting a plant DNA sample by sampling, and simultaneously inputting the collected plant DNA sample and an anticoagulant into a sampling test tube arranged on an oscillator, so that the plant DNA sample and the anticoagulant are fully mixed under the action of oscillation of the oscillator;
s102, centrifuging an uncondensed plant DNA sample by a centrifuge, sucking out the centrifuged nuclear cell mixed solution by a suction pipe, and transferring the nuclear cell mixed solution into a sampling test tube with a culture solution for storage;
s103, centrifuging the nuclear cell mixed solution in the sampling test tube by using a centrifuge, removing supernatant, and repeating the steps for five times in sequence to obtain pure nuclear cells;
s104, directly placing the frozen nuclear cell sample block into a mortar of a grinder, adding liquid nitrogen, and quickly grinding;
s105, placing the uniformly ground sample into a centrifuge tube of a homogenizer, adding an RNA extraction reagent into the centrifuge tube, carrying out vacuum treatment on the centrifuge tube, and carrying out homogenization treatment on the mixed solution in the centrifuge tube through the homogenizer;
s106, separating and extracting RNA from the homogenized mixed solution; eluting the separated RNA by an oscillator; the determination of the RNA concentration was accomplished using a spectrophotometer.
As shown in fig. 3, in step S106 in the embodiment of the present application, the method for separating and extracting RNA is specifically as follows:
s201: filling the homogenized mixed solution into a centrifuge tube, adding chloroform into the centrifuge tube, fully oscillating and uniformly mixing in an oscillator, and then standing for 15min at room temperature;
s202: centrifuging at 4deg.C under the action of 12000g centrifugal force for 15min, collecting supernatant, transferring to new centrifuge tube, adding isopropanol, mixing, and standing at room temperature;
s203: centrifuging at 4deg.C under the action of 12000g centrifugal force for 15min, and discarding supernatant after centrifuging to obtain precipitated RNA.
In step S202 of the embodiment of the application, the standing time at room temperature is 10-15min.
As shown in fig. 4, in step S106 in the embodiment of the present application, the method for eluting RNA is specifically as follows:
s301: adding 75% ethanol into the precipitated RNA centrifuge tube, and oscillating the centrifuge tube with low power to suspend and precipitate RNA in the centrifuge tube;
s302: the suspension and sediment centrifuge tube at 4 ℃,8000g of centrifugal force, remove the supernatant, and dry at room temperature.
In S302 of the embodiment of the application, the centrifugation time for suspension sedimentation is 15-20min.
As shown in fig. 5, in step S106 in the embodiment of the present application, the method for detecting RNA concentration using a spectrophotometer is specifically as follows:
s401: adding 0.5-2 mu l of ultrapure water into a detection eye by using a pipetting gun, resetting a signal receiver, and operating an "Ok" key of operation software to enable the instrument to enter an initialized state;
s402: loading a control sample, and running an operation software 'Blank' key;
s403: and loading the eluted RNA, and running an operation software 'Measure' key, wherein the concentration of the RNA is instantly displayed on a display screen.
As shown in fig. 6, in step S101 in the embodiment of the present application, the preparation method of the plant DNA sample includes:
s501, taking 5.0 g of young leaves of plants, putting the young leaves into liquid nitrogen, freezing and grinding the young leaves into powder;
s502, transferring the mixture into 12ml of an extracting solution preheated at 65 ℃ for 20 minutes, and then adding chloroform with the same volume for uniform mixing;
s503, oscillating for 15 minutes at normal temperature and low speed, centrifuging for 15 minutes at 12000 r/min, taking supernatant, and adding 2/3 volume of isopropanol to precipitate DNA;
s504, taking out flocculent DNA, washing twice with 70% ethanol, naturally drying, adding 500 μl of pH8.0TE buffer for dissolution and quantification for later use.
While the application has been described with respect to what is presently considered to be the most practical and preferred embodiments, it is to be understood that the application is not limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications, equivalents, and alternatives falling within the spirit and scope of the application.
Claims (10)
1. The utility model provides a collection device suitable for molecular marker detects, its characterized in that, the collection device suitable for molecular marker detects specifically includes:
the sample extraction module is connected with the central control module and comprises a sampling needle and a sampling test tube, and is used for collecting plant DNA samples prepared in advance through sampling, and the collected plant DNA samples and an anticoagulant are simultaneously input into the sampling test tube on the oscillator;
the nuclear cell sample collection module is connected with the central control module and comprises a centrifuge and a straw, the centrifugal processing is carried out on the uncoagulated plant DNA sample through the centrifuge, the centrifuged nuclear cell mixed solution is sucked out through the straw, and the nuclear cell mixed solution is transferred into a sampling test tube with a culture solution for storage;
the nuclear cell sample cleaning and storing module is connected with the central control module and comprises a centrifugal machine, the centrifugal machine is used for carrying out centrifugal treatment on the nuclear cell mixed solution in the sampling test tube, the supernatant is removed, and the steps are repeated for five times in sequence to obtain pure nuclear cells;
the sample grinding treatment module is connected with the central control module and comprises a grinder, frozen nuclear cell sample blocks are directly placed into a mortar of the grinder, liquid nitrogen is added into the mortar, and the mortar is quickly ground;
the sample homogenizing treatment module is connected with the central control module and comprises a homogenizer, a uniformly ground sample is placed in a centrifuge tube of the homogenizer, an RNA extraction reagent is added into the centrifuge tube, the centrifuge tube is subjected to vacuum treatment, and the mixed solution in the centrifuge tube is subjected to homogenizing treatment through the homogenizer;
the separation and extraction module is connected with the central control module and comprises a centrifugal machine, and is used for separating and extracting RNA from the homogenized mixed solution;
the RNA eluting module is connected with the central control module and comprises an oscillator and a centrifugal machine, and is used for eluting the separated RNA through the oscillator;
the RNA marker detection module is connected with the central control module and comprises a spectrophotometer, and the spectrophotometer is used for measuring the concentration of RNA;
the central control module is connected with the sample extraction module, the nuclear cell sample collection module, the nuclear cell sample cleaning and storage module, the sample grinding and processing module, the sample homogenizing and processing module, the separation and extraction module, the RNA eluting module, the RNA marker detection module and the power supply module and is used for controlling the sample extraction module to collect and anticoagulate a plant DNA sample, the nuclear cell sample collection module to collect nuclear cells, the nuclear cell sample cleaning and storage module to clean the nuclear cells and other impurities and store the nuclear cells in a freezing way, the sample grinding and processing module and the sample homogenizing and processing module to grind the samples in a homogenizing way, and the RNA eluting module and the RNA marker detection module to elute the separated RNA and measure the concentration of the RNA;
the power supply module is connected with the sample extraction module, the nuclear cell sample collection module, the nuclear cell sample cleaning and storing module, the sample grinding and processing module, the sample homogenizing and processing module, the separation and extraction module, the RNA eluting module, the RNA marker detection module and the central control module and is used for providing power support for the normal operation of instruments or equipment in each module.
2. The collection device for molecular marker detection according to claim 1, wherein the rotational speed of centrifugation of the plant DNA sample in the nuclear cell sample collection module is 1500rpm for 25min.
3. The collection device for molecular marker detection according to claim 1, wherein the rotational speed of the nuclear cell sample washing and storing module for centrifuging the nuclear cell mixture is 1200rpm for 20min.
4. The collection device for molecular marker detection according to claim 1, wherein the homogenization temperature in the sample homogenization treatment module is 10-25 ℃ for 5-10min.
5. A collection method suitable for molecular marker detection based on the collection device suitable for molecular marker detection according to any one of claims 1-4, characterized in that the collection method suitable for molecular marker detection comprises the following specific steps:
firstly, collecting plant DNA samples by sampling, simultaneously inputting the collected plant DNA samples and an anticoagulant into a sampling test tube arranged on an oscillator, and fully mixing the plant DNA samples and the anticoagulant under the action of oscillation of the oscillator;
centrifuging the uncondensed plant DNA sample by a centrifuge, sucking out the centrifuged nuclear cell mixed solution by a suction pipe, and transferring the nuclear cell mixed solution into a sampling test tube with a culture solution for storage;
step three, centrifuging the nuclear cell mixed solution in the sampling test tube by using a centrifuge, removing supernatant, and repeating the steps for five times in sequence to obtain pure nuclear cells;
step four, directly placing the frozen nuclear cell sample block into a mortar of a grinder, adding liquid nitrogen, and quickly grinding;
placing the uniformly ground sample into a centrifuge tube of a homogenizer, adding an RNA extraction reagent into the centrifuge tube, carrying out vacuum treatment on the centrifuge tube, and carrying out homogenization treatment on the mixed solution in the centrifuge tube through the homogenizer;
step six, separating and extracting RNA from the homogenized mixed solution; eluting the separated RNA by an oscillator; the determination of the RNA concentration was accomplished using a spectrophotometer.
6. The method of claim 5, wherein in step one, the method of preparing a plant DNA sample comprises:
s1, taking 5.0 g of young leaves of plants, putting the young leaves into liquid nitrogen, freezing and grinding the young leaves into powder;
s2, transferring the mixture into 12ml of an extracting solution preheated at 65 ℃ for 20 minutes, and then adding chloroform with the same volume for uniform mixing;
s3, oscillating for 15 minutes at normal temperature and low speed, centrifuging for 15 minutes at 12000 r/min, taking supernatant, and adding 2/3 volume of isopropanol to precipitate DNA;
s4, taking out flocculent DNA, washing the flocculent DNA twice with 70% ethanol, naturally drying the flocculent DNA, and adding 500 μl of buffer solution with pH of 8.0TE for dissolution and quantification for later use.
7. The method of claim 5, wherein in step six, the isolating and extracting RNA specifically comprises:
firstly, filling the homogenized mixed solution into a centrifuge tube, adding chloroform into the centrifuge tube, fully oscillating and uniformly mixing in an oscillator, and then standing for 15min at room temperature;
secondly, centrifuging for 15min under the action of a centrifugal force of 12000g at the temperature of 4 ℃, sucking supernatant fluid after the centrifugation is finished, transferring the supernatant fluid into a new centrifuge tube, adding isopropanol, uniformly mixing, and standing at room temperature;
thirdly, centrifuging for 15min under the action of 12000g of centrifugal force at 4 ℃, and discarding supernatant after the centrifugation is finished to obtain the RNA after precipitation.
8. The method according to claim 7, wherein the second step is performed at room temperature for a period of 10 to 15 minutes.
9. The method of claim 5, wherein in step six, the elution of RNA comprises:
(1) Adding 75% ethanol into the precipitated RNA centrifuge tube, and oscillating the centrifuge tube with low power to suspend and precipitate RNA in the centrifuge tube;
(2) Centrifuging the suspension precipitate in a centrifuge tube at 4deg.C under the action of 8000g centrifugal force for 15-20min, removing supernatant, and air drying at room temperature.
10. The method of claim 5, wherein in step six, the method of using a spectrophotometer to perform the determination of the concentration of RNA comprises:
1) Adding 0.5-2 mu l of ultrapure water into a detection eye by using a pipetting gun, resetting a signal receiver, and operating an "Ok" key of operation software to enable the instrument to enter an initialized state;
2) Loading a control sample, and running an operation software 'Blank' key;
3) And loading the eluted RNA, and running an operation software 'Measure' key, wherein the concentration of the RNA is instantly displayed on a display screen.
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