CN111440790A - Method for simultaneously extracting DNA and RNA of salivary spots - Google Patents
Method for simultaneously extracting DNA and RNA of salivary spots Download PDFInfo
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Abstract
The invention discloses a method for simultaneously extracting DNA and RNA of salivary spots. The invention provides a method for synchronously extracting DNA and RNA from on-site salivary spots, which comprises the following steps of 1) soaking the on-site salivary spots with carriers in a reaction system containing DTT and cell dissociation buffer solution, and carrying out incubation reaction to obtain reaction products; 2) removing the carrier in the reaction product, collecting the reaction product from which the carrier is removed, then enabling the reaction product from which the carrier is removed to flow through a DNA adsorption column, and collecting the adsorption column adsorbed with DNA and the column passing liquid containing RNA; 3) purifying the column passing liquid containing RNA and the adsorption column adsorbed with DNA respectively to obtain RNA and DNA; the method can be used for nucleic acid extraction of common on-site test materials such as oral swabs, cigarette butts, water cup wipers and the like, and the obtained DNA and RNA samples can obtain ideal detection results in downstream STR typing and fluorescent quantitative PCR detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for simultaneously extracting DNA and RNA of salivary spots.
Background
The salivary stains are one of the most common biological material evidence types at the scene of various case events, and comprise oral swab samples, and also comprise cigarette butts, tableware wipes, bites and other trace field test materials possibly adhered with oral epithelial cells, wherein nucleic acid information such as DNA, RNA and the like is contained in the oral swab samples. With the development of DNA inspection technology, compared with other biological evidences such as exfoliated cells and the like, the salivary spot is easier to obtain DNA STR typing with comparative value through DNA analysis, and plays an irreplaceable important role in case detection and suspect investigation. Meanwhile, on the basis of DNA STR typing detection, the tissue sources of the on-site biological detection materials such as saliva spots and the like are deduced by means of technical means such as RNA analysis and the like, so that accurate information can be provided for comprehensive analysis and case process reconstruction of case-related biological material evidence, and the evidence value of the case-related biological material evidence is further improved.
However, with the current trend of criminal forms and criminal techniques, on-site minute quantity of old biological evidence has become a major subject of inspection and research in the field of forensic science, and plays an important role in case practice. Due to low nucleic acid content and high nucleic acid damage degree of trace and old biological material evidence, the nucleic acid sample which not only meets the DNA STR test but also accords with the RNA analysis is difficult to be extracted and obtained simultaneously by applying the prior art method. In practice, due to technical condition limitation, one more urgent analysis content has to be selected in DNA and RNA analysis, and a great deal of important information is lost. Therefore, a method for simultaneously extracting DNA and RNA which can be applied to on-site salivary spots is needed.
Research shows that in the field of basic medicine and biological research, mature DNA and RNA simultaneous extraction kits or methods are developed. However, these methods are only suitable for large amounts of clean culture samples such as cells and tissues, and when applied to field biological examination materials with trace amount of on-site cases, complex inhibitors or impurity components, DNA and RNA nucleic acid samples meeting the downstream detection requirements are difficult to obtain, and the practical requirements of simultaneous extraction and analysis of trace salivary spot DNA and RNA on-site cases in criminal technology cannot be met.
Disclosure of Invention
An object of the present invention is to provide a method for simultaneous extraction of DNA and RNA from in situ salivary plaques.
The method provided by the invention comprises the following steps:
1) soaking the spot saliva spots with the carriers in a reaction system containing DTT and cell dissociation buffer solution, and incubating for reaction to obtain reaction products;
2) removing the carrier in the reaction product, collecting the reaction product with the carrier removed, then flowing the reaction product with the carrier removed through a DNA adsorption Column (specifically DNeasy Spin Column in the AllPrep DNA/RNA Mini Kit in the embodiment of the invention), and collecting the adsorption Column with the adsorbed DNA and the Column passing liquid containing RNA;
3) purifying the column passing liquid containing RNA and the adsorption column adsorbed with DNA respectively to obtain RNA and DNA;
the purification of the RNA-containing column-passing liquid is carried out according to a method comprising the following steps:
3) -1, passing the RNA-containing Column-passing liquid through an RNA adsorption Column (in the embodiment of the present invention, specifically RNeasy Spin Column in AllPrep DNA/RNA Mini Kit), to obtain an adsorption Column on which RNA is adsorbed;
3) 2, passing an RNA elution buffer (in the embodiment of the invention, RNase-free water is specifically adopted as the RNA elution buffer) through the adsorption column adsorbed with the RNA, standing for 5-10min at room temperature (18-25 ℃), centrifuging (centrifuging at 16200g for 1min), and collecting the first column passing liquid to obtain the RNA;
the DNA purification is carried out according to a method comprising the following steps:
3) a, using DNA elution Buffer solution (EB Buffer or water or other DNA eluent can be used as the DNA elution Buffer solution, which can be carried by the AllPrep DNA/RNA Mini Kit, or prepared or purchased) to flow through the adsorption column adsorbed with the DNA, standing for 8-15min at 60-65 ℃ (the standing time can be 10min specifically), and centrifugally collecting the first column passing solution to obtain the DNA.
In the above method, the method for purifying the RNA-containing column-passing liquid further comprises the following steps between steps 3) -1 and 3) -2: and washing the adsorption column adsorbed with the RNA to obtain the washed adsorption column adsorbed with the RNA.
The washing is specifically divided into the following:
① mu.l Buffer RW1 (Wash) was added to the Column RNeasy Spin Column with adsorbed RNA and centrifuged at 13000rpm for 15 s;
② adding 500. mu.l buffer RPE (washing) to the RNA-adsorbed Column RNeasy Spin Column after the previous step, and centrifuging at 13000rpm for 15 s;
note that: buffer RPE should be used with the appropriate volume of absolute ethanol.
③ adding 500 μ l buffer RPE (washing) to the RNA-adsorbed Column RNeasy Spin Column after the previous step, and centrifuging at 13000rpm for 2 min;
④ 13000 centrifuging at 13000rpm for 1min, repeating twice, ensuring no liquid residue on RNeasy Spin Column;
⑤ transferring the RNA-adsorbed column to clean 2m L EP tube without RNase or DNase, and centrifuging at 13000rpm for 1 min;
in the above method, the method for purifying the RNA-containing column-passing liquid further comprises, before passing through the RNA adsorption column in step 3) -1, the steps of: and adding an equal volume of 70-80% ethanol aqueous solution into the RNA-containing column-passing liquid to obtain a mixed solution.
In the above method, in the method for purifying the RNA-containing column-passing liquid, in 3) -2, after the first column-passing liquid is collected, the first column-passing liquid is passed through the adsorption column with the RNA adsorbed thereon again, and is allowed to stand at room temperature for 5-10min, and the second column-passing liquid is collected by centrifugation; and combining the first column solution and the second column solution to obtain RNA.
In the method for purifying DNA, before passing through the adsorption column with adsorbed DNA with DNA elution buffer, the following steps are included: and washing the adsorption column adsorbed with the DNA to obtain the adsorption column adsorbed with the DNA after washing.
In the above-mentioned method, the first step of the method,
the washing is as follows:
① to the DNA-adsorbed adsorption Column DNeasy Spin Column obtained in 2) above, 500. mu.l of BufferAW1 was added (washed), and centrifuged at 13000rpm for 15 s;
note that: buffer AW1 should be added with an appropriate volume of absolute ethanol before use.
② adding 500. mu.l of BufferAW2 (washing) to the DNA-adsorbed adsorption Column DNeasy Spin Column after the previous treatment, and centrifuging at 13000rpm for 2 min;
③ transferring the DNA-adsorbed Column DNeasy spin Column after the above treatment to a new RNase-free and DNase-free clean 2m L EP tube, and centrifuging at 13000rpm for 1 min;
④ 13000 centrifuging at 13000rpm for 1min, repeating twice, ensuring no liquid residue on DNeasy Spin Column;
in the above method, in the method for purifying DNA, in 3) -a, after the first column-passing liquid is collected, the first column-passing liquid flows through the adsorption column on which the DNA is adsorbed after washing, stands for 8-15min at 60-65 ℃, and is centrifuged to collect the second column-passing liquid; and combining the first column solution and the second column solution to obtain DNA.
In the method, the concentration of the DTT in the reaction system is 1-2mM, the concentration of the DTT in the reaction system is specifically 1.6 mM. in the embodiment of the invention, the reaction system containing the DTT and the cell dissociation Buffer solution is obtained by uniformly mixing the DTT and the cell dissociation Buffer solution, and in the embodiment of the invention, the cell dissociation Buffer solution is specifically Buffer R L T in an AllPrep DNA/RNA Mini Kit.
In the method, in the step 1), the reaction condition is that the incubation is carried out for more than or equal to 1.5 hours at room temperature; the reaction condition is specifically incubation for 2.5h at 18-25 ℃.
In the above method, the RNA elution buffer is RNase-free water.
In the above method, the on-site salivary spot with carrier is a trace on-site detection material such as oral swab, cigarette butt or cup wipe.
In step 2), the step of removing the carrier from the reaction product and collecting the reaction product with the carrier removed is performed according to the following method: clamping the carrier from the reaction product by using clean ophthalmic tweezers without RNase and DNase, placing the carrier in a disposable injector without RNase and DNase, pressing a piston of the injector tightly to ensure that residual liquid on the carrier is extruded and flows out completely, recovering the residual liquid on the carrier, recovering the residual liquid into an original centrifugal tube, and uniformly mixing the residual liquid with the liquid in the reaction product except the carrier to obtain the reaction product (liquid) without the carrier;
before the reaction product of the carrier removal passes through a DNA adsorption column, the method also comprises the following steps: centrifuging the reaction product with the carrier removed at 13000rpm for 2min (removing common small particle impurities such as dust, mud and the like on the on-site material checking carrier), and taking a centrifugal supernatant;
the collection is to pass the supernatant through a DNA adsorption column at 13000rpm for 30s, repeat the centrifugation for 2 times until no liquid exists in the adsorption column, and collect the adsorption column with the adsorbed DNA and the column passing liquid containing RNA.
The DNA adsorption Column may be specifically AllPrep DNASpin Column in AllPrep DNA/RNA Mini Kit.
The DNA and RNA synchronous extraction method established by the research can be used for extracting nucleic acid of common on-site test materials such as oral swabs, cigarette butts, water cup wipers and the like, and the obtained DNA and RNA samples obtain relatively ideal detection results in downstream STR typing and fluorescent quantitative PCR detection, so that the operation is simple and convenient, the results are stable, the requirements of actual case handling are met, and the method can be used for analyzing and detecting DNA and RNA of on-site salivary spots.
The establishment of the method is based on the optimization of a finished kit method, the kit only suitable for a large number of clean samples such as cells, tissues and the like is applied to a method for simultaneously extracting the salivary spot DNA and RNA of a field inspection material with trace field and complex inhibitors of common cases such as oral swabs, cigarette butts, water cup wipers and the like, the possibility is provided for the synchronous analysis of the DNA and the RNA of the field inspection material, the effect is expected to be played in case-related biological evidence inspection, more comprehensive information is provided for case detection, suspect carving and case process reconstruction, and the evidence value of the field salivary spot is further improved. However, the cost of the finished product kit is high, and in the future popularization and application, an extraction method with low cost should be further developed, and the application effect of the kit in the detection materials such as blood, exfoliated cells and the like is investigated according to the actual needs.
The method of the invention has the following advantages:
1. carrier (cell) treatment, which is to adopt operation steps different from cell and tissue suspension in carrier digestion and lysis, directly soak the oral epithelial cells attached to the surfaces of cotton swabs and cigarette butts and the carrier in lysis solution Buffer R L T, and to avoid the loss of cells or nucleic acid fragments dissociated due to cell disruption, the carrier is only sheared into fragments with proper size to form a larger surface area so as to be beneficial to the permeation of the lysis solution, thereby obtaining higher digestion efficiency;
2. digestion and lysis, namely using DTT (dithiothreitol) with lower toxicity and stronger reducibility instead of using β -ME (2-mercaptoethanol) as a reducing agent to destroy protein disulfide bonds, preventing DNA from forming dimers while destroying the protein disulfide bonds, being beneficial to dissociating from the surface of a complex carrier to obtain oral epithelial cells, and simultaneously being capable of destroying the cells more efficiently under the condition of impurities and even inhibitors, thereby obtaining a nucleic acid sample and being more beneficial to obtaining a satisfactory detection result in subsequent detection;
3. nucleic acid purification: after the nucleic acid purification step is completed, increasing the centrifugation times, and removing the purification buffer solution cleanly so as to avoid the reduction of the elution efficiency caused by the excessively strong binding force between the nucleic acid and the purification column due to the existence of the purification buffer solution in the subsequent nucleic acid elution, and simultaneously avoid the adverse effect of a buffer system on the nucleic acid detection;
4.① adding eluent, adding room temperature standing elution step, standing for 10 minutes at room temperature, prolonging elution time properly, dissolving RNA in eluent more fully, not degrading RNA due to too long standing time, ② increasing elution temperature to 65 deg.C in elution, prolonging incubation time to ten minutes, increasing elution temperature, reducing binding force between DNA and centrifugal column, increasing DNA solubility, prolonging elution time, facilitating DNA to dissolve fully, improving elution efficiency, changing elution conditions, increasing nucleic acid extraction rate, and obtaining nucleic acid in limited micro-detection material to a greater extent, thereby obtaining more real reaction sample condition in subsequent detection.
5. The DNA and RNA synchronous extraction method established by the research can be used for extracting nucleic acid of common on-site test materials such as oral swabs, cigarette butts, water cup wipers and the like, and the obtained DNA and RNA samples obtain relatively ideal detection results in downstream STR typing and fluorescent quantitative PCR detection, so that the operation is simple and convenient, the results are stable, the requirements of actual case handling are met, and the method can be used for analyzing and detecting DNA and RNA of on-site salivary spots. The establishment of the method is based on the optimization of a finished kit method, changes the key steps of carrier treatment, cell lysis, nucleic acid elution and the like by relying on a centrifugal column system of the kit, introduces a DNA and RNA nucleic acid extraction method applied to basic research into forensic nucleic acid inspection, applies the kit which is only suitable for a large amount of clean and cultured samples such as cells, tissues and the like to field biological inspection materials with trace amount of cases and complex inhibitors, provides possibility for the synchronous analysis of DNA and RNA of the case field case-related biological inspection materials, and can further examine the application effect of the method in the field biological inspection materials such as blood, exfoliated cells and the like.
Drawings
FIG. 1 shows the results of oral swab STR typing tests extracted by the methods described in the specification.
Figure 2 is a graph of the detection results of STR typing of cigarette butt samples extracted by the method of the specification.
FIG. 3 shows the detection results of STR typing of water cup wipes extracted by the method of the specification.
FIG. 4 shows the result of the detection of STR typing of oral swabs extracted by the optimization method.
FIG. 5 shows the result of STR typing detection of a cigarette butt sample extracted by the optimization method.
FIG. 6 shows the detection result of STR typing of the water cup wipes extracted by the optimization method.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The apparatus and reagents used in the following examples are as follows:
1) the required instruments for the experiment were as follows:
2) the main reagents are as follows:
AllPrep DNA/RNA Mini Kit Germany QIAGEN
GlobalFilerTMPCR Amplification Kit, Thermo corporation, USA
Example 1 method for synchronously extracting DNA and RNA from salivary spots
Method for synchronously extracting DNA and RNA of salivary spots
1. Experimental sample preparation (the following three types all belong to saliva stains on common case sites in criminal technical work practice and are also the most important biological material evidence on case sites):
(1) oral swab: the cotton swab is rolled to wipe the side wall of the mouth of the person to be detected.
(2) Cigarette end: fresh cigarette butt samples.
(3) Water cup wiping: wiping the saliva spots at the cup mouth of the water cup by a dry-wet wiping method.
2. Comparative example: extraction of DNA and RNA from AllPrep DNA/RNA Mini Kit
Extracting DNA and RNA of the sample 1 by using the existing AllPrep DNA/RNA Mini Kit, and the operating steps of the instruction:
1) simultaneous obtaining of DNA and RNA by lysis
(1) Cutting appropriate amount of carrier (cotton swab wiping part or outer paper skin of cigarette end filter) into pieces with appropriate size, placing into clean 1.5m L centrifuge tube without RNase and DNase, and adding 600 μ l Buffer R L T and 6 μ l β -ME;
(2) clamping the carrier by clean ophthalmological forceps without RNase and DNase, placing the carrier in a disposable injector without RNase and DNase, pressing a piston of the injector tightly to ensure that residual liquid on the carrier is extruded and flows out completely, and recycling the residual liquid into an original centrifugal tube to remove the carrier;
(3) centrifuging at 13000rpm for 2min (removing common small particle impurities such as dust, mud and the like on a field material detection carrier), taking the supernatant, passing through an AllPrep DNA Spin Column adsorption Column (13000rpm, 30s, repeatedly centrifuging for 2 times until no liquid exists in the adsorption Column), placing the adsorption Column in a clean 2m L EP tube without RNase and DNase, and storing at 4 ℃ (the AllPrep DNAsin Column adsorption Column is used for DNA purification, and the Column passing liquid is used for RNA purification);
2) and (3) purifying RNA:
① adding 70% ethanol into the Column-passing liquid, centrifuging 700 μ l, passing through RNeasy Spin Column (13000rpm, 15s), and repeating the operation until all liquid is transferred;
② Add 700. mu.l Buffer RW1 to RNeasy Spin Column, centrifuge at 13000rpm for 15 s;
③ Add 500. mu.l Buffer RPE to RNeasy Spin Column, centrifuge at 13000rpm for 15 s;
note that: buffer RPE should be used with the appropriate volume of absolute ethanol.
④ Add 500. mu.l Buffer RPE to RNeasy Spin Column, centrifuge at 13000rpm for 2 min;
note that: the procedure can be repeated to ensure no liquid residue on RNeasy Spin Column.
⑤ the RNeasy Spin Column was transferred to a new RNase-free, DNase-free clean 2m L EP tube and centrifuged at 13000rpm for 1 min;
⑥ placing RNeasy Spin Column in a clean 1.5m L centrifuge tube without RNase or DNase, adding 35 μ l RNase-free water, centrifuging at 13000rpm for 1min, and collecting supernatant as RNA;
⑦ the obtained RNA was stored at-80 ℃ until use.
And (3) purifying DNA:
① Add 500. mu.l Buffer AW1 to DNeasy Spin Column, centrifuge at 13000rpm for 15 s;
note that: buffer AW1 should be added with an appropriate volume of absolute ethanol before use.
② Add 500. mu.l Buffer AW2 to RNeasy Spin Column, centrifuge at 13000rpm for 2 min;
③ the RNeasy Spin Column was transferred to a new RNase-free, DNase-free clean 2m L EP tube and centrifuged at 13000rpm for 1 min;
note that: the procedure was repeated to ensure that no liquid remained on the DNeasy Spin Column.
④ placing DNeasy Spin Column in a clean 1.5m L centrifuge tube without RNase or DNase, adding 100 μ l EB Buffer, standing at room temperature for 1min, centrifuging at 13000rpm for 1min, and repeating the operation twice;
⑤ the obtained DNA was stored at-80 ℃ until use.
3. The invention relates to a method for synchronously extracting DNA and RNA of salivary spots
The research is based on the existing AllPrep DNA/RNA Mini Kit as a description, the steps of cracking, adsorption, elution and the like are optimized, and the DNA and RNA synchronous extraction method which can be used for on-site material detection and trace nucleic acid samples is finally formed.
1) Simultaneous obtaining of DNA and RNA by lysis
(1) Shearing a proper amount of a carrier (a cotton swab wiping part or outer paper skin of a cigarette butt filter) into fragments with proper sizes, placing the fragments into a clean 1.5m L centrifugal tube without RNase and DNase, adding 600 mu l of Buffer R L T (dissociated cells) and 20 mu l of DTT (disrupted membrane lysed cells, wherein the final concentration of the cells in a reaction system is 1.6mM), fully shaking and uniformly mixing, and incubating at room temperature (25 ℃) for 2.5 hours to obtain a reaction product;
(2) clamping the carrier from the reaction product by using clean ophthalmic tweezers without RNase and DNase, placing the carrier into a disposable injector without RNase and DNase, pressing a piston of the injector tightly to ensure that residual liquid on the carrier is extruded and flows out completely, recovering the residual liquid on the carrier, recovering the residual liquid into an original centrifugal tube, and uniformly mixing the residual liquid with the liquid in the reaction product except the carrier to obtain the reaction product without the carrier;
(3) centrifuging the reaction product with the carrier removed at 13000rpm for 2min (removing common dust, soil and other small particle impurities on the on-site test material carrier), taking the supernatant, passing through an AllPrep DNA Spin Column (13000rpm, 30s, repeatedly centrifuging for 2 times until no liquid exists in the Column), and collecting the Column-passing liquid containing RNA and the adsorption Column with DNA.
The AllPrep DNA Spin Column was used for DNA purification, and the Column-passing liquid was used for RNA purification.
The column was placed in RNase-free, DNase-free clean 2m L EP tube and stored at 4 ℃.
2) And (3) purifying RNA:
① adding 70% ethanol water solution (removing protein, fatty acid, etc. and helping nucleic acid to bind to RNA adsorption column) with equal volume into the RNA-containing column liquid obtained in 1), centrifuging 700 μ l, passing through RNeasy spin column (RNA adsorption column, 13000rpm, 15s), and repeating the operation until all liquid is transferred to obtain RNA-adsorbed adsorption column;
② mu.l Buffer RW1 (Wash) was added to the Column RNeasy Spin Column with adsorbed RNA and centrifuged at 13000rpm for 15 s;
③ adding 500. mu.l buffer RPE (washing) to the RNA-adsorbed Column RNeasy Spin Column after the previous step, and centrifuging at 13000rpm for 15 s;
note that: buffer RPE should be used with the appropriate volume of absolute ethanol.
④ adding 500 μ l buffer RPE (washing) to the RNA-adsorbed Column RNeasy Spin Column after the previous step, and centrifuging at 13000rpm for 2 min;
⑤ 13000 centrifuging at 13000rpm for 1min, repeating twice, ensuring no liquid residue on RNeasy Spin Column;
⑥ transferring the RNA-adsorbed column to clean 2m L EP tube without RNase or DNase, and centrifuging at 13000rpm for 1 min;
⑦ placing the adsorption column with RNA adsorbed thereon in a clean 1.5m L centrifuge tube without RNase and DNase, adding 35 μ l RNase-free water, standing at room temperature (25 deg.C) for 10min, centrifuging at 13000rpm (16200 g centrifugal force) for 1min, and collecting the first centrifugation column-passing solution;
passing the column solution after the first centrifugation through an adsorption column adsorbing RNA in the step again, standing at room temperature for 10min, centrifuging at 13000rpm (16200 g centrifugal force) for 1min, and collecting the column solution after the second centrifugation;
and combining the first column solution and the second column solution to obtain RNA.
⑧ the obtained RNA was stored at-80 ℃ until use.
And (3) purifying DNA:
① to the DNA-adsorbed adsorption Column DNeasy Spin Column obtained in the above 1), 500. mu.l of BufferAW1 was added (washed), and centrifuged at 13000rpm for 15 s;
note that: buffer AW1 should be added with an appropriate volume of absolute ethanol before use.
② adding 500. mu.l of BufferAW2 (washing) to the DNA-adsorbed adsorption Column DNeasy Spin Column after the previous treatment, and centrifuging at 13000rpm for 2 min;
③ transferring the DNA-adsorbed Column DNeasy Spin Column after the above treatment to a new RNase-free and DNase-free clean 2m L EP tube, and centrifuging at 13000rpm for 1 min;
④ 13000 centrifuging at 13000rpm for 1min, repeating twice, ensuring no liquid residue on DNeasy Spin Column;
⑤ placing the adsorption Column DNeasy Spin Column with DNA adsorbed in the previous step into a clean 1.5m L centrifuge tube without RNase and DNase, adding 100 μ l EB Buffer (provided by the kit), standing at 65 deg.C for 10min, centrifuging at 13000rpm for 1min, and collecting the first centrifugation Column-passing liquid;
passing the column solution after the first centrifugation through an adsorption column adsorbing DNA in the step again, standing at 65 ℃ for 10min, centrifuging at 13000rpm (16200 g of centrifugal force) for 1min, and collecting the column solution after the second centrifugation;
and combining the first column solution and the second column solution to obtain the DNA.
⑥ the obtained DNA was stored at-80 ℃ until use.
Second, detecting
1. Nucleic acid concentration and OD value
The DNA concentration and OD value were measured separately using a Nanodrop2000 spectrophotometer.
Concentration and OD of extracted DNA260/280The values are shown in tables 1 and 2, and compared with the optimization method, the DNA OD extracted by the kit method260/280Both are larger than 2, which indicates that the RNA removal is not thorough and the DNA extraction quality is low.
Table 1 shows the concentration and OD value of DNA extracted in comparative example (detection in kit)
Table 2 shows the concentration and OD value of DNA extracted by the method of the present invention
2. DNA STR typing
Extracting DNA by the above two different methods with GlobalfillerTMAnd amplifying the PCR Amplification Kit, sequencing by using an ABI3500xl sequencer, and parting the DNA STR.
Partial results of oral swabs, butts and cup wipes taken from the kit instructions are shown in figures 1-3, and it can be seen that no effective STR typing was obtained.
The partial results of all DNA extracted by the method of the invention are shown in figures 4-6, and it can be seen that the DNA samples all obtain correct STR typing results, and the peak graphs are clear, sharp and balanced, thus meeting the requirements of DNA STR detection and comparison.
The results of the oral swabs, butts and cup wipes extracted according to the kit instructions and the method of the invention are summarized in table 3, the kit method only detects 2-4 loci in the oral swab sample, and has no comparison value, and no other samples are detected. The method detects all loci from all samples, has correct STR typing result and clear, sharp and balanced peak pattern, and meets the requirements of DNA STR detection and comparison.
TABLE 3 STR typing results for each set of sample DNA
3. Reverse transcription fluorescence quantitative PCR detection of β -actin mRNA and 18S rRNA signals in RNA
1) Reverse transcription to obtain cDNA
Invitrogen was usedTMSuperScriptTMIII First-Strand Synthesis Supermix for qRT-PCR kit (Thermo Fisher U.S.A., 2 × RT Reaction Mix in this kit contains RandomPrimers, Oligo dT Primers) the RNA obtained above was reverse transcribed to synthesize cDNA as follows:
(1) reaction system (20 μ l):
(2) reaction procedure:
after the reaction is finished, the reaction product is collected to obtain cDNA.
Taking a proper amount of cDNA template, carrying out PCR amplification according to the following reaction system and reaction program, and carrying out parallel amplification twice on each sample.
2) Fluorescent quantitative PCR
The principle is as follows:
using the TaqMan probe method, the probe and the primer are
18S GE Assays and ACTB GE Assays of the Gene Expression Assays series (Thermo Fisher USA). The PCR reagent is
FastAdvanced Master Mix (Thermo Fisher USA). The reaction was performed on a Step One quantitative PCR instrument.
(1) PCR reaction (20. mu.l):
(2) PCR amplification procedure:
(3) data processing and analysis:
after the PCR reaction is finished, CT values of each reaction are obtained, and the average CT value of the expression of the two genes of the 18S rRNA and β -Actin (ACTB) mRNA of each sample and the ratio of the CT values of the two genes are calculated.
The 18S rRNA and β -actin mRNA signal detection results are shown in tables 4 and 5. the Ct values of the 18S rRNA and β -actin mRNA of the RNA sample extracted according to the kit specification are close to the threshold value, and β -actin mRNA detection signals are not obtained from a plurality of samples (variance statistics is not carried out on the group where the samples are located), so that the detection requirements cannot be met.
TABLE 4 comparative example (kit test) extraction of 18S rRNA and β -actin mRNA Ct values of RNA samples
TABLE 5 extraction of 18S rRNA and β -actin mRNA Ct values of RNA samples by the method of the present invention
Claims (10)
1. A method for synchronously extracting DNA and RNA from a spot saliva stain comprises the following steps:
1) soaking the spot saliva spots with the carriers in a reaction system containing DTT and cell dissociation buffer solution, and incubating for reaction to obtain reaction products;
2) removing the carrier in the reaction product, collecting the reaction product with the carrier removed, passing the reaction product with the carrier removed through a DNA adsorption column, and collecting the adsorption column adsorbed with DNA and the column passing liquid containing RNA;
3) purifying the column passing liquid containing RNA and the adsorption column adsorbed with DNA respectively to obtain RNA and DNA;
the purification of the RNA-containing column-passing liquid is carried out according to a method comprising the following steps:
3) -1, flowing the RNA-containing column-passing liquid through an RNA adsorption column to obtain the adsorption column adsorbed with RNA;
3) 2, flowing RNA elution buffer solution through the adsorption column adsorbed with RNA, standing at room temperature for 5-10min, and centrifuging to collect first column passing solution to obtain RNA;
the DNA purification is carried out according to a method comprising the following steps:
3) and a, using a DNA elution buffer solution to flow through the adsorption column adsorbed with the DNA, standing for 8-15min at 60-65 ℃, and centrifugally collecting the first column passing solution to obtain the DNA.
2. The method of claim 1, wherein:
the method for purifying the RNA-containing column-passing liquid further comprises the following steps between the steps 3) -1 and 3) -2: and washing the adsorption column adsorbed with the RNA to obtain the washed adsorption column adsorbed with the RNA.
3. The method of claim 2, wherein:
in the method for purifying the RNA-containing column-passing liquid, the method further comprises the following steps before flowing through the RNA adsorption column in the step 3) -1: and adding an equal volume of 70-80% ethanol aqueous solution into the RNA-containing column-passing liquid to obtain a mixed solution.
4. A method according to claim 2 or 3, characterized in that:
in the method for purifying the column passing liquid containing the RNA, in 3) -2, after the first column passing liquid is collected, the first column passing liquid flows through the adsorption column adsorbed with the RNA again, stands for 5-10min at room temperature, and is centrifuged to collect the second column passing liquid; and combining the first column solution and the second column solution to obtain RNA.
5. The method according to any one of claims 1-4, wherein:
in the method for purifying DNA, before passing through the adsorption column with adsorbed DNA with DNA elution buffer, the following steps are included: and washing the adsorption column adsorbed with the DNA to obtain the adsorption column adsorbed with the DNA after washing.
6. The method according to any one of claims 1-5, wherein:
in the method for purifying the DNA, in 3) -a, after the first column-passing liquid is collected, the first column-passing liquid flows through the adsorption column which is adsorbed with the DNA after washing, stands for 8-15min at 60-65 ℃, and is centrifuged to collect the second column-passing liquid; and combining the first column solution and the second column solution to obtain DNA.
7. The method according to any one of claims 1-6, wherein: the concentration of the DTT in the reaction system is 1-2 mM.
8. The method according to any one of claims 1-7, wherein: in the step 1), the reaction condition is that the incubation is carried out for more than or equal to 1.5 hours at room temperature.
9. The method according to any one of claims 1-8, wherein: the RNA elution buffer is RNase-free water.
10. The method according to any one of claims 1-9, wherein:
the on-site salivary spot with carrier is a mouth swab, a cigarette butt or a cup wipe.
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