A method for simultaneously extracting DNA, RNA and protein from saliva stains

Abstract

The present invention provides a method for simultaneously extracting DNA, RNA and protein from a saliva stain, and belongs to the field of biotechnology. The steps of the method of the present invention include: 1) placing the saliva stain in a reaction system containing a lysis buffer to obtain a lysis product; 2) centrifuging the lysis product, collecting the lysis supernatant, and passing it through a DNA adsorption column and an RNA adsorption column in sequence, collecting the adsorption column adsorbed with DNA and the adsorption column adsorbed with RNA, and finally the column liquid flowing through the RNA adsorption column; 3) purifying the adsorption column containing DNA, the adsorption column containing RNA and the filtrate passing through the RNA adsorption column for the first time, respectively, to obtain a DNA solution, an RNA solution and a protein solution. The test results show that the obtained DNA, RNA and protein samples can be subjected to STR typing, fluorescent quantitative PCR, and ELISA salivary amylase to detect samples of corresponding concentrations, and the minimum amount of saliva used is 10 μL.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for simultaneously extracting DNA, RNA and protein from saliva spots. Background Art

[0002] Saliva stains are one of the most common types of biological evidence at the scene of various crimes, which contain information such as DNA, RNA, and proteins. Individual identification and kinship inference can be achieved through DNA analysis. RNA analysis can provide effective evidence for the age of an individual and the nature of body fluid stains. Proteins are the main components of all biological evidence and are usually embedded in the matrix of other biological molecules, such as polynucleotides, lipids, carbohydrates, and small molecules. The proteins in the sample reflect the transcription and translation programs of the original cell type. Since the development of mass spectrometry, proteomics technology has extracted more information from the protein components of physical evidence, providing strong evidence for sample identification and recognition. At present, after all DNA in a large sample is degraded, it is still possible to detect and generate peptide maps of genetic variations from proteins. However, there is currently a lack of multi-omics analysis methods for trace evidence.

[0003] In the actual case handling, on-site biological evidence (blood, saliva, hair, secretions, etc.) has become an important basis for providing intelligence. However, biological objects such as saliva and secretions are rare, and due to technical limitations, it is often necessary to choose between DNA STR testing, RNA analysis, and protein analysis, which results in the loss of a large amount of important information. At present, the technology for extracting DNA, RNA, or protein from trace biological evidence is relatively mature, but there is no relevant report on the technology for extracting the three components at the same time. Summary of the invention

[0004] The object of the present invention is to provide a method for simultaneously extracting DNA, RNA and protein from a salivary spot. The method provided by the present invention can effectively and accurately extract DNA, RNA and protein from 10 μL of salivary spots at the same time, and the minimum amount of saliva contained in the salivary spot is 10 μL.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for simultaneously extracting DNA, RNA and protein from a salivary stain, which is characterized by comprising the following steps: (1) mixing the salivary stain with a lysate and shaking to obtain a lysate; (2) centrifuging the lysate for 10 to 20 minutes, taking the supernatant of the lysate, adding it to a DNA adsorption column, centrifuging the DNA adsorption column, and collecting the supernatant from which the DNA is removed; performing a DNA purification treatment on the DNA adsorption column after the centrifugation to obtain a DNA solution; (3) mixing the supernatant from which the DNA is removed with anhydrous ethanol, then adding it to an RNA adsorption column, centrifuging the RNA adsorption column, and collecting the supernatant from which the RNA is removed; performing an RNA purification treatment on the RNA adsorption column after the centrifugation to obtain an RNA solution; and (4) mixing the supernatant from which the RNA is removed with a buffer solution APP and centrifuging, removing the supernatant and adding ethanol to centrifuge to obtain a protein precipitate, and dissolving the protein precipitate in PBS to obtain a protein solution.

[0007] Preferably, the saliva stain in step (1) needs to be cut into pieces ≤ 0.5 cm 2 .

[0008] Preferably, the centrifugal DNA adsorption column in step (2) is centrifuged at 10,000 to 15,000 rpm for 20 to 40 seconds.

[0009] Preferably, the DNA purification process comprises the following steps: adding inhibitor removal solution IR to the centrifuged DNA adsorption column in step (2), centrifuging and discarding the supernatant, adding rinse solution WB, centrifuging and discarding the supernatant; taking the centrifuged DNA adsorption column, adding 20 to 80 μL elution buffer EB, centrifuging and collecting the supernatant containing DNA, and retaining the DNA adsorption column; re-adding the supernatant containing DNA to the retained DNA adsorption column, centrifuging, and obtaining a DNA solution.

[0010] Preferably, the amount of anhydrous ethanol added in step (3) is 1 to 1.5 times the amount of the supernatant from which DNA is removed.

[0011] Preferably, the RNA purification treatment comprises the following steps: washing the RNA adsorption column after centrifugation in step (3) with a rinse solution, centrifuging to obtain a rinsed RNA adsorption column, adding 5 to 15 μL RNase free water, centrifuging after standing at room temperature, retaining the RNA adsorption column, and obtaining a supernatant containing RNA; re-adding the supernatant containing RNA to the retained RNA adsorption column, standing at room temperature, and centrifuging to obtain an RNA solution.

[0012] Preferably, before adding RNase free water, the RNA adsorption column after rinsing and centrifugation is first dried.

[0013] Preferably, the amount of the RNA-containing supernatant added again is 8 to 12 μL.

[0014] Preferably, the ethanol in step (4) is a 60-80% ethanol aqueous solution, and the amount of ethanol added is 0.3-0.7 ml.

[0015] Preferably, the amount of PBS solution added in step (4) is 80 to 120 μL.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The method for extracting DNA, RNA and protein simultaneously established by the present invention can be used for the simultaneous extraction of DNA, RNA and protein from trace saliva spots on site, with a minimum usage of 10 μL. It can also simultaneously realize personal identification and saliva spot property identification based on STR typing, fluorescent quantitative PCR and ELISA salivary amylase.

[0018] The method of the invention is simple and easy to implement, and the detection result is stable, which lays a foundation for realizing multi-omics analysis of trace evidence in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The DNA STR typing results extracted by the method of the present invention.

[0020] Figure 2 Comparative analysis of DNA STR typing results extracted using the method.

[0021] Figure 3 Standard curve of human salivary amylase alpha 1. DETAILED DESCRIPTION

[0022] The present invention provides a method for simultaneously extracting DNA, RNA and protein from a salivary stain, comprising the following steps: (1) mixing the salivary stain with a lysate to obtain a lysate; (2) centrifuging the lysate for 10 to 20 minutes, taking the supernatant of the lysate, adding a DNA adsorption column, centrifuging the DNA adsorption column, collecting the supernatant from which DNA is removed; subjecting the centrifuged DNA adsorption column to DNA purification to obtain a DNA solution; (3) mixing the supernatant from which DNA is removed with anhydrous ethanol, then adding an RNA adsorption column, centrifuging the RNA adsorption column, collecting the supernatant from which RNA is removed; subjecting the centrifuged RNA adsorption column to RNA purification to obtain an RNA solution. (4) mixing the supernatant from which RNA is removed with a buffer APP and centrifuging, adding ethanol after removing the supernatant and centrifuging to obtain a protein precipitate, dissolving the protein precipitate in PBS to obtain a protein solution. The minimum dosage of the salivary stain of the present invention is 10 μL. The salivary stain of the present invention may originate from a crime scene. The limitation of the centrifugation time of the present invention can increase the lysis efficiency and increase the DNA extraction efficiency.

[0023] In the present invention, the saliva stain in step (1) needs to be cut into pieces ≤ 0.5 cm2 The amount of the lysate is 500-700 μL, preferably 600 μL. The lysate of the present invention is preferably lysate RLT Plus. After adding the lysate in step (1) of the present invention, the mixture needs to be allowed to stand for 3-7 minutes, preferably 5 minutes; the shaking time is preferably 30 minutes.

[0024] In the present invention, the centrifugation speed of the lysate in step (2) is 10000-15000 rpm, preferably 13000 rpm; the centrifugation condition of the DNA adsorption column is 10000-15000 rpm for 20-40 s, preferably 13000 rpm for 30 s. The DNA of the sample is adsorbed on the membrane of the DNA adsorption column after centrifugation.

[0025] In the present invention, the DNA purification treatment comprises the following steps: adding inhibitor removal solution IR to the centrifuged DNA adsorption column in step (2), centrifuging and discarding the supernatant, adding rinsing solution WB, centrifuging and discarding the supernatant; taking the centrifuged DNA adsorption column, adding 20-80 μL elution buffer EB, centrifuging and collecting the supernatant containing DNA, and retaining the DNA adsorption column; re-adding the supernatant containing DNA to the retained DNA adsorption column, centrifuging, and obtaining a DNA solution. The amount of inhibitor removal solution IR added in the present invention is 400-600 μL, preferably 500 μL. The present invention uses the rinsing solution WB to clean the DNA adsorption column twice, the amount of the first rinsing solution added is 700 μL, and centrifuged at 12000 rpm for 30 seconds; the amount of the second rinsing solution added is 500 μL, and centrifuged at 12000 rpm for 30 seconds. The present invention takes the centrifuged DNA adsorption column after rinsing and puts it into an empty collection tube, centrifuges at 13000 rpm for 2 minutes, and removes the rinsing solution as much as possible to prevent the residual ethanol in the rinsing solution from inhibiting the downstream reaction. The elution buffer EB of the present invention needs to be added to the middle part of the DNA adsorption column membrane. The elution buffer EB is preheated in a water bath at 65-70° C. for better effect.

[0026] In the present invention, the amount of anhydrous ethanol added in step (3) is 1 to 1.5 times, preferably 1.25 times, of the supernatant from which DNA is removed. In the present invention, precipitation may occur after the addition of anhydrous ethanol, but it does not affect the extraction process, and can be immediately mixed by blowing. The amount of the mixed solution of the supernatant from which DNA is removed and anhydrous ethanol used in the present invention needs to be less than 700 μL, and if the amount is large, it can be used in multiple times. The condition of the centrifugal RNA adsorption column of the present invention is centrifugation at 13000 rpm for 30 seconds.

[0027] In the present invention, the RNA purification treatment comprises the following steps: washing the RNA adsorption column after centrifugation in step (3) with a rinse solution, centrifuging and discarding the supernatant, adding 5 to 15 μL RNase free water, centrifuging after standing at room temperature, retaining the RNA adsorption column, and obtaining a supernatant containing RNA; re-adding the RNA-containing supernatant to the retained RNA adsorption column, centrifuging after standing at room temperature, and obtaining an RNA solution. The RNA adsorption column of the present invention is washed with a rinse solution (Wash Solution) 3 times, adding 700 μL Wash Solution 1 for the first time, and adding 500 μL Wash Solution 2 and Wash Solution 3 for the second and third times, respectively, and the centrifugation conditions are all 12000 rpm centrifugation for 30s. The present invention puts the rinsed RNA adsorption column back into an empty collection tube, centrifuges at 13000 rpm for 2 minutes, and removes the rinse solution as much as possible to prevent the residual ethanol in the rinse solution from inhibiting the downstream reaction. Before adding RNase free water in the present invention, the RNA adsorption column after rinsing and centrifugation is placed at room temperature for 30 minutes, or placed on a clean bench for ventilation for 15 minutes to fully dry. The drying step of the present invention can improve the extraction efficiency and purity of RNA. The room temperature placement time of the present invention is 3 to 7 minutes, preferably 5 minutes. The centrifugation condition after adding RNase free water in the present invention is 14800rpm centrifugation for 10 minutes, and the centrifugation condition after re-adding the retained RNA adsorption column is 14800rpm centrifugation for 1 minute. The amount of RNA-containing supernatant re-added in the present invention is 8 to 12 μL, preferably 10 μL.

[0028] In the present invention, the amount of buffer APP added in step (4) is equal to the volume of the supernatant from which DNA is removed; after the supernatant from which DNA is removed is mixed with anhydrous ethanol, it is necessary to vortex and oscillate to mix, place at room temperature for 15 minutes to precipitate protein, and then centrifuge at 13000rpm for 5-10 minutes, and pour out as much supernatant as possible to remove the supernatant. The ethanol in step (4) of the present invention is a 60-80% ethanol aqueous solution, and the amount of ethanol added is 0.3-0.7ml; after the ethanol is added, it is necessary to invert and centrifuge for 1 minute to remove the supernatant, and try to clean the residual liquid with a pipette. The present invention requires that the protein precipitate obtained by centrifugation be dried at room temperature for 5-10 minutes to allow the ethanol to evaporate. The amount of PBS solution added in step (4) of the present invention is 80-120μL, preferably 100μL. The present invention requires that the protein solution be stored at -20°C.

[0029] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0030] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 Method for simultaneous extraction of DNA, RNA and protein from saliva stains

[0032] 1. Sample processing

[0033] 1.1 Cut the saliva stain sample into small pieces no larger than 0.5 cm 2 Transfer to a 1.5 ml centrifuge tube containing 600 μL tissue lysis buffer RLTPlus, let stand for 5 minutes, and shake vigorously on an oscillator for 30 minutes to fully lyse.

[0034] 1.2 Centrifuge the homogenized lysate at 13,000 rpm for 15 min, take the supernatant and add it to the DNA adsorption column.

[0035] 1.3 Centrifuge the DNA adsorption column at 13000 rpm for 30 seconds and retain the supernatant, which contains miRNA, RNA and protein. The genomic DNA is adsorbed on the membrane of the DNA adsorption column after centrifugation.

[0036] 1.4 Use a micropipette to accurately estimate the volume of the supernatant and add 1.25 times the volume of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Mix it by blowing immediately without centrifugation. Immediately add the mixture (less than 700 μL each time, and can be added twice at most) to an RNA adsorption column RA (the adsorption column is placed in a collection tube) and centrifuge at 13000 rpm for 30 seconds. Keep the supernatant after removing RNA for protein extraction.

[0037] 2. DNA purification steps:

[0038] 2.1 Add 500 μL inhibitor removal solution IR to the DNA adsorption column in step 1.3, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0039] 2.2 Add 700 μl of rinse solution WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

[0040] 2.3 Add 500 μL of washing buffer WB, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0041] 2.4 Place the centrifuged DNA adsorption column back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes. Remove the rinse solution as much as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions.

[0042] 2.5 Take out the DNA adsorption column after centrifugation, put it into a clean centrifuge tube, add 50μL elution buffer EB to the middle part of the adsorption membrane (preheating the elution buffer in a 65-70℃ water bath is better), leave it at room temperature for 5 minutes, centrifuge it at 12000rpm for 1 minute, keep the DNA adsorption column, and collect the DNA supernatant. Add the obtained DNA supernatant back to the retained DNA adsorption column, leave it at room temperature for 2 minutes, centrifuge it at 12000rpm for 1 minute, and obtain the DNA solution for subsequent reactions.

[0043] 3. RNA purification steps:

[0044] 3.1 Add 700 μL Wash Solution 1, 12,000 μL to the RNA adsorption column centrifuged in step 1.4.

[0045] Centrifuge at rpm for 30 s and discard the waste liquid.

[0046] 3.2 Add 500μL Wash Solution 2 / 3, centrifuge at 12000mpm for 30s, discard the waste liquid. Add 500μL Wash Solution 2 / 3, repeat.

[0047] 3.3 Place the adsorption column RA back into the empty collection tube and centrifuge at 13000rpm for 2min to remove the rinse solution as much as possible to prevent the residual ethanol in the rinse solution from inhibiting the downstream reaction. After centrifugation, place the adsorption column at room temperature for 30min, or place it on a clean bench for ventilation for 15min to fully dry.

[0048] 3.4 Take out the adsorption column RA and put it into an RNase free centrifuge tube. Add 10μL RNase free water in the middle part of the adsorption membrane according to the expected RNA yield, leave it at room temperature for 5 minutes, and centrifuge it at 14800 rpm for 10 minutes.

[0049] 3.5 Add 10 μL of the solution obtained after centrifugation in 3.4 back into the RNA adsorption column RA in 3.4, place at room temperature for 5 minutes, and centrifuge at 14800 rpm for 1 minute to obtain the RNA solution, which can be used immediately for downstream reactions or stored in low temperature as soon as possible.

[0050] 4. Protein Purification

[0051] 4.1 Add an equal volume of buffer APP to the supernatant from which RNA was removed in step 1.4, vortex and mix well, and leave at room temperature for 15 minutes to precipitate the protein.

[0052] 4.2 Centrifuge at 13000rpm for 5-10min, pour out as much supernatant as possible to remove the supernatant. Add 0.5ml of 70% ethanol, invert and centrifuge for 1min, remove the supernatant, and use a pipette to remove as much residual liquid as possible to obtain protein precipitation.

[0053] 4.3 Dry the protein precipitate at room temperature for 5-10 minutes to allow the ethanol to evaporate.

[0054] 4.4 Dissolve the dried protein precipitate in 100 μL PBS solution to obtain the protein solution, and store it at -20°C for later use.

[0055] Comparative Example 1

[0056] The difference from the steps of Example 1 is:

[0057] In step 1.1, the saliva stains were not cut into pieces.

[0058] Centrifuge in step 1.2 for 3 minutes.

[0059] The amount of elution buffer EB added in step 2.5 is 100 μL.

[0060] Remove the drying process of the RNA adsorption column after centrifugation in step 3.3.

[0061] In 3.4, add 30-50 μL of RNase free water, place at room temperature for 1 min, and centrifuge at 12,000 rpm for 1 min.

[0062] Remove the steps in 3.5.

[0063] Example 2

[0064] 1. STR typing of DNA extracted from saliva spots

[0065] 1.1 The extracted DNA was amplified by PCR, and the amplification system was configured to be 10 μL ( DNA identity identification system Y-Plus kit purchased from Basepoint Cognitive Technology Co., Ltd.): 2.5 μL 4×PCR reaction premix VII, 2.0 μL 5×23plex primer mixture, 5.5 μL DNA. PCR amplification program: pre-deformation: 95°C, 2 min; 27 cycles: 94°C, 5 s, 60°C, 2 min; final extension: 60°C, 10 min; low temperature storage: 15°C.

[0066] 1.2 Electrophoresis detection (3130 electrophoresis analyzer)

[0067] (1) Prepare the sample loading system according to the ratio of internal standard: deionized formamide = 0.5 μL: 9.5 μL, and dispense 10 μL / well;

[0068] (2) Step 1.1 PCR product usage: 1 μL, Allelic Ladder usage: 1 μL;

[0069] (3) After denaturation at 95°C for 3 min, quickly cool to 0°C for 3 min;

[0070] (4) Electrophoresis detection: injection voltage 1.2 kVolts, injection time 24 s.

[0071] 2. Detection of RNA extracted from saliva spots

[0072] 2.1 Reverse transcription reaction to detect RNA (miRcute enhanced miRNA cDNA first-strand synthesis kit (KR211): The extracted RNA was reverse transcribed, and the reverse transcription reaction system was 20 μL: 10 μL 2×miRNARTReactionBuffer, 2 μL miRNART Enzyme Mix. Reverse transcription reaction procedure: 42°C, 60 min; 95°C, 5 min; 4°C, 5 min. The reverse transcription product was divided and stored at -20°C.

[0073] 2.2 PCR reaction (miRcute enhanced miRNA fluorescence quantitative detection kit (SYBR Green) (FP411)): Take an appropriate amount of the reverse transcription product obtained in step 2.1 for PCR amplification to obtain PCR amplification products. PCR amplification reaction system 20μL: 10μL 2×miRcute Plus miRNAPreMix (SYBR&ROX); 0.4μL Forward Primer (hsa-miR-203a-3p detection primer and hsa-miR-205-5p detection primer were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.); 0.4μL Reverse Primer; 2μL miRNA first-chain cDNA; 7.2μL ddH2O. PCR amplification program (completed by fluorescence quantitative PCR instrument): initial denaturation: 95°C, 15 min; 5 cycles: 94°C, 20 s, 64°C, 30 s, 72°C, 34 s; 40 cycles: 94°C, 20 s, 60°C, 34 s.

[0074] 2.3 Calculate the Ct values ​​of the two salivary plaque miRNA markers (miR203-3p and miR205-5p) of the samples.

[0075] 3. Detection of proteins extracted from salivary plaques (human salivary amylase α1 (AMY1) ELISA kit)

[0076] 3.1 Set up standard wells, blank wells and sample wells respectively. Add 100μL of the diluted standard to the standard wells, 100μL of the standard to the blank wells, and 100μL of the extracted protein diluent to the remaining wells (it is recommended that all samples and standards to be tested be set up in duplicate wells during the test). Cover the ELISA plate with a film and incubate at 37℃ for 90min. Tips: When adding samples, add the sample to the bottom of the ELISA plate, try not to touch the wall of the well, and gently shake to mix and avoid bubbles. The addition time should be controlled within 10min.

[0077] 3.2 Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour.

[0078] 3.3 Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1min, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Tip: Here and other washing steps can use a plate washer (refer to the parameter settings of Beijing Top DEM-3 plate washer: 2-point aspiration, add 350μL of washing solution to each well, vibrate the plate for 5s, and aspirate for 0.5s). Please proceed to the next step immediately after washing the plate, and do not let the microplate dry.

[0079] 3.4 Add 100 μL of enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 min.

[0080] 3.5 Shake off the liquid in the wells and wash the plate 5 times using the same method as step 3.

[0081] 3.6 Add 90 μL of substrate solution (TMB) to each well, cover the plate with film, and incubate at 37°C in the dark for about 15 minutes. Tips: shorten or extend the time as appropriate according to the actual color development, but do not exceed 30 minutes. When a clear gradient appears in the standard well (clear blue gradient appears in the first 4 color development wells), it can be terminated. Turn on the microplate reader 15 minutes in advance to preheat.

[0082] 3.7 Add 50 μL of stop solution to each well to terminate the reaction. Tip: The order of adding the stop solution should be the same as that of adding the substrate solution.

[0083] 3.8 Immediately measure the optical density (OD value) of each well using a microplate reader at a wavelength of 450 nm.

[0084] Example 3 Application of the extraction method of the present invention

[0085] 1. Saliva plaque preparation for DNA, RNA, and protein co-extraction

[0086] 10 μL of fresh human saliva was taken on the blood card using a pipette and dried for one day to prepare a saliva spot sample. The steps of the experimental group were as follows: The implementation steps were the same as those in Example 1. The steps of the control group were as follows: The implementation steps were the same as those in the comparative example.

[0087] 2. Extracted DNA testing

[0088] The detection method is the same as step 1 in Example 2. Figure 1 and Figure 2 It can be seen that 23 STR loci can be analyzed in the 10 μL saliva sample of the experimental group: DYS549, DYS19, DYS392, DYS643, DYS447, DYS557, DYS388, DYS570, DYS635, DYS448, DYS393, DYS398I, DYS390, DYS389II, DYS438, DYS458, DYS481, DYS449, DYS596, DYS518, DYF387S1, DYS593, DYS522. In the control group, only one STR locus was analyzed in the 10 μL saliva sample of the experimental group: DYS19.

[0089] 3. Extracted RNA Detection

[0090] The concentration of extracted RNA was detected using the nanodrop detection method. The results are shown in Table 1.

[0091] Table 1 Comparison of RNA concentrations extracted from saliva spots by different extraction methods

[0092]

[0093]

[0094] The results of detecting the extracted RNA using the qRT-PCR method described in step 2 of Example 2 are shown in Table 2.

[0095] Table 2 Comparison of CT values ​​of RNA extracted from saliva spots by qRT-PCR detection using different methods

[0096]

[0097] As shown in Tables 1 and 2, the RNA extracted by the extraction method of the present invention has a high concentration; the extracted RNA can be successfully detected using microRNA specific markers: miR-203-3p, miR205-5p (CT value ≤ 30), and the improved method can improve the detection rate.

[0098] 4. Detection of extracted proteins

[0099] The detection steps are the same as step 3 of Example 2, and the standard curve of the determination is as follows: Figure 3 The OD values ​​of the test samples are shown in Table 3. The concentrations of human salivary amylase α1 in the samples were calculated by the standard curve and the measured sample OD values ​​and are shown in Table 4.

[0100] Table 3 OD values ​​of proteins extracted from saliva spots

[0101]

[0102]

[0103] Table 4 Concentration of proteins extracted from salivary plaques

[0104] sample Protein (human salivary amylase α1) concentration (ng / mL) Sample 1 1.27593 Sample 2 1.13278 Blank control 1.10422

[0105] Table 3-4 and Figure 3 (Standard curve) The results show that the extraction method of the present invention can extract protein (human salivary amylase α1) in 10 μL of salivary plaque with good stability.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously extracting DNA, RNA and protein from salivary stains, It is characterized in that The steps include: (1) mixing the saliva stain with the lysis solution and shaking to obtain a lysate; (2) centrifuging the lysate at 13,000 rpm for 10 to 20 minutes, taking the supernatant of the lysate, adding it to a DNA adsorption column, centrifuging the DNA adsorption column, and collecting the supernatant after removing the DNA; performing DNA purification on the centrifuged DNA adsorption column to obtain a DNA solution; (3) mixing the DNA-removed supernatant with anhydrous ethanol, then adding it to an RNA adsorption column, centrifuging the RNA adsorption column, and collecting the RNA-removed supernatant; performing RNA purification on the RNA adsorption column after centrifugation to obtain an RNA solution; (4) mixing the RNA-removed supernatant with buffer APP and centrifuging, removing the supernatant and adding ethanol, centrifuging to obtain a protein precipitate, and dissolving the protein precipitate in PBS to obtain a protein solution; The saliva stain in step (1) needs to be cut into pieces ≤ 0.5 cm 2 ; The centrifugal DNA adsorption column in step (2) is centrifuged at 10,000 to 15,000 rpm for 20 to 40 seconds; The DNA purification process comprises the following steps: adding inhibitor removal solution IR to the centrifuged DNA adsorption column in step (2), centrifuging and discarding the supernatant, adding rinse solution WB, centrifuging and discarding the supernatant; taking the centrifuged DNA adsorption column, adding 20 to 80 μL elution buffer EB, centrifuging and collecting the supernatant containing DNA, and retaining the DNA adsorption column; adding the supernatant containing DNA back to the retained DNA adsorption column, centrifuging, and obtaining a DNA solution; The amount of anhydrous ethanol added in step (3) is 1 to 1.5 times the amount of the supernatant from which DNA is removed; The RNA purification process comprises the following steps: washing the RNA adsorption column after centrifugation in step (3) with a rinse solution, centrifuging to obtain the rinsed RNA adsorption column, adding 5 to 15 μL of RNase free water, leaving it at room temperature and then centrifuging, retaining the RNA adsorption column to obtain a supernatant containing RNA; re-adding the supernatant containing RNA to the retained RNA adsorption column, leaving it at room temperature and then centrifuging to obtain an RNA solution; Before adding RNase free water, dry the RNA adsorption column after rinsing and centrifugation; The amount of the RNA-containing supernatant added again is 8 to 12 μL; The ethanol in step (4) is a 60-80% ethanol aqueous solution, and the amount of ethanol added is 0.3-0.7 mL; The amount of PBS solution added in step (4) is 80 to 120 μL.

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