CN113151398A - Method for detecting nucleic acid molecules in exosomes - Google Patents
Method for detecting nucleic acid molecules in exosomes Download PDFInfo
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Abstract
The invention relates to a method for simply, conveniently and rapidly detecting nucleic acid molecules in exosomes, which comprises the steps of cracking exosomes in a sample and releasing exosome nucleic acid molecules; performing reverse transcription reaction, wherein the exosome is subjected to reverse transcription reaction, namely RT-For-All, without extracting nucleic acid molecules or after extracting nucleic acid molecules after cracking, so that All RNAs in the exosome are subjected to reverse transcription in the same reaction; wherein a reverse transcription buffer comprising a universal adaptor (e.g., a miRNA Adapter) and a random primer is used in the reverse transcription reaction. The invention also provides a kit for detecting nucleic acid molecules in exosomes. The method releases the content components by directly dissolving the exosome, does not need to extract RNA and DNA, and directly carries out reverse transcription of all RNA in the same reverse transcription reaction. Various RNAs and DNAs can be detected from trace exosomes.
Description
Technical Field
The invention relates to a method for simply, conveniently and rapidly detecting the characteristics of exosomes, and mainly relates to the detection of exosome inclusion RNA and exosome inclusion DNA.
Background
Exosomes (Exosomes) are extracellular vesicles of about 30-150nm in size, with lipid bilayer membrane structures. Exosomes were found in sheep reticulocytes as early as 1983, but have been considered as a way for cells to excrete waste. With the intensive research on exosomes in recent years, the exosomes are found to carry a large amount of important information such as protein, lipid, DNA and RNA (miRNA, mRNA, lncRNA and circRNA) and participate in intercellular communication and information exchange, and play an important role in multiple aspects such as antigen presentation, tumor growth and migration, tissue injury repair and the like according to the cell types from which the exosomes are derived.
Almost all cells in the human body secrete exosomes such as immune cells, endothelial cells, tumor cells and the like under normal or pathological conditions. There is also a certain difference in the functional properties of the active substances contained in exosomes of different cell sources, and when the exosomes are secreted by a host cell and delivered to a recipient cell, the function and physiological state of the recipient cell are mainly regulated or changed by the active substances contained in the exosomes. Due to its stable physical properties and relatively sophisticated data support, among others, exosome RNAs, in particular mirnas, are of great interest. Research in recent years proves that exosome miRNA has a very close relationship with diseases, such as participation in immune diseases, viral infection, neurodegenerative disease transmission, cancer occurrence and metastasis and the like, and can be used as a biomarker for disease diagnosis, so that detection of inclusion RNA is very important for understanding and researching functional characteristics of exosome.
The extraction of exosome RNA or DNA which is commonly used at present is mostly based on phenol extraction or chromatographic column, the method can not only cause the loss of partial RNA or DNA molecules and directly influence the subsequent result analysis, but also is relatively complex and time-consuming. In particular, the amount of each exosome sample is generally small, and it is not easy to extract and quantify RNA or DNA.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting nucleic acid molecules in exosomes, and particularly relates to a method for simply, rapidly and conveniently detecting exosome nucleic acid molecules by one-step method. The method of the invention releases the content components by directly dissolving the exosomes without extracting RNA and DNA, and directly carries out reverse transcription of all RNA in the same reverse transcription reaction by the optimized proportion of the contents of primers (i.e. universal joints and random primers) and enzymes (i.e. reverse transcriptase and PolyA polymerase). Various RNAs and DNAs can be detected from trace exosomes. The detection method can be used for quickly detecting the RNA and DNA of the exosome in cell culture supernatant, plasma, urine or other body fluids and other samples.
Specifically, the invention provides the following technical scheme:
1. a method for detecting nucleic acid molecules in exosomes, comprising the steps of:
lysing exosomes in the sample for releasing exosome nucleic acid molecules; preferably, the sample is a cell culture fluid, plasma, urine or other bodily fluid sample;
performing reverse transcription reaction, wherein the exosome is subjected to reverse transcription reaction, namely RT-For-All, without extracting nucleic acid molecules or after extracting nucleic acid molecules after cracking, so that All RNAs in the exosome are subjected to reverse transcription in the same reaction;
wherein a reverse transcription buffer comprising a universal adaptor (e.g., miRNA Adapter) and a random primer is used in the reverse transcription reaction;
preferably, the reverse transcription reaction is followed by quantitative PCR or sequencing detection.
2. The detection method according to item 1, wherein the nucleic acid molecule comprises RNA (e.g., mRNA, miRNA, IncRNA, cirRNA) and DNA (e.g., cDNA).
3. The detection method according to item 1, wherein in the step of lysing the exosomes, lysis is performed using an exosome lysis buffer, wherein the lysis buffer comprises a pH buffer salt (preferably Tris-Cl), BSA, an RNase inhibitor and a non-ionic detergent;
preferably, the non-ionic detergent is TritonX-100, Tween20, NP-40, Igepal in any one or more combinations.
4. The assay of item 1 wherein the lysis buffer further comprises proteinase K for inhibiting nuclease activity and protecting nucleic acids from degradation; preferably, the lysis buffer further comprises DTT.
5. The detection method according to item 1, wherein a reverse transcription reaction system comprising a reverse transcription buffer, a reverse transcriptase, and a PolyA polymerase, preferably further comprising dNTP and ATP, is used in the reverse transcription reaction step.
6. The detection method according to item 1, wherein the conditions of the reverse transcription reaction include 37 ℃ to 42 ℃ for 5 to 30 min.
7. The detection method according to item 1, wherein a product of RT-For-All reverse transcription reaction can be directly used as a substrate For various detections, For example, For nucleic acid detection by quantitative PCR (qPCR); can also be used as a library construct for nucleic acid NGS.
8. A kit for detecting nucleic acid molecules in exosomes, comprising a reverse transcription reaction system; wherein the reverse transcription reaction system comprises a reverse transcription buffer solution, a reverse transcriptase and a PolyA polymerase, and preferably also comprises dNTP and ATP;
wherein the reverse transcription buffer comprises a universal linker (e.g., miRNA Adapter) for reverse transcription of small RNA molecules and a random primer for reverse transcription of large RNA molecules.
9. The kit of clause 8, further comprising a lysis buffer, wherein the lysis buffer comprises a pH buffering salt (preferably Tris-Cl), BSA, an RNase inhibitor, and a nonionic detergent;
preferably, the non-ionic detergent is TritonX-100, Tween20, NP-40, Igepal in any one or more combinations.
10. The kit of clause 9, wherein the lysis buffer further comprises proteinase K for inhibiting nuclease activity and protecting nucleic acids from degradation; preferably, the lysis buffer further comprises DTT.
In a preferred embodiment of the present invention, in the reverse transcription reaction system, the concentration of the reverse transcriptase is 1 to 10U/. mu.L of the reaction system; more preferably, the concentration of the reverse transcriptase may be 3-8U/. mu.L reaction system, 4-7U/. mu.L reaction system, 4-8U/. mu.L reaction system, 4-9U/. mu.L reaction system, 4-10U/. mu.L reaction system, 5-10U/. mu.L reaction system, 6-10U/. mu.L reaction system, 7-10U/. mu.L reaction system, 8-10U/. mu.L reaction system, or 9-10U/. mu.L reaction system.
In a preferred embodiment of the present invention, the concentration of PolyA polymerase in the reverse transcription reaction system is 0.05 to 0.5U/. mu.l reaction system; more preferably, the concentration of the PolyA polymerase may be 0.1 to 0.5U/. mu.L reaction system, 0.2 to 0.5U/. mu.L reaction system, 0.3 to 0.5U/. mu.L reaction system, or 0.4 to 0.5U/. mu.L reaction system.
In a preferred embodiment of the present invention, the concentration of the universal linker in the reverse transcription buffer is 0.2-2. mu.M; more preferably, the concentration of the universal primer may be 0.2-0.8. mu.M, 0.2-0.7. mu.M, 0.2-0.5. mu.M, 0.3-0.8. mu.M, 0.3-0.6. mu.M, 0.3-0.5. mu.M, 0.4-0.8. mu.M, 0.4-0.6. mu.M, 0.4-0.5. mu.M, 0.5-0.8. mu.M, 0.5-1.5. mu.M, 0.8-1.5. mu.M; most preferably 0.5. mu.M.
In a preferred embodiment of the present invention, the concentration of the random primer in the reverse transcription buffer is 0.2 to 5. mu.M; more preferably, the concentration of the random primer may be 0.2-5. mu.M, 0.2-4. mu.M, 0.2-3. mu.M, 0.2-2. mu.M, 0.2-1. mu.M, 0.2-0.8. mu.M, 0.2-0.6. mu.M, 0.2-0.5. mu.M, 0.2-0.4. mu.M, 0.2-0.3. mu.M, 0.2-2. mu.M, 0.2-3. mu.M, 1-2. mu.M, 1-3. mu.M, or 2-3. mu.M; most preferably 2. mu.M.
More specifically, the invention provides the following technical scheme:
(1) exosome extraction
Collecting 0.5-10mL cell culture solution, plasma, urine or other body fluid samples, adding into a centrifuge tube, centrifuging at 500 Xg for 10min to remove cell debris, transferring the supernatant into a new centrifuge tube, and filtering the supernatant with 0.22 μ M or 0.45 μ M PES filter membrane according to the size of exosome or other cell secretory vesicles to be detected so as to further remove the cell debris; then adding 1/3 volume of ExoSere exosome purification reagent (GeneCopoeia, Inc, USA) into the filtered supernatant, fully mixing uniformly, and then placing at 4 ℃ for overnight; the sample was then centrifuged at 6,000rpm for 15min, and the supernatant carefully aspirated, taking care not to touch the exosome pellet; the exosome suspension was resuspended in 10-200. mu.L of 1 XPBS buffer.
(2) Cleavage of exosomes
And (2) taking 5 mu L of the exosome suspension obtained in the step (1), adding 5 mu L of LExoCt lysis buffer solution, uniformly mixing, placing on ice for 5min, then placing at 75 ℃ for 10min, and placing on ice after the reaction is finished.
The composition of the ExoCt lysis buffer is as follows:
Tris-Cl pH buffer salts: 10-100mM
b.BSA：1-100μg/mL
RNase inhibitor: 0.1-10U/. mu.L
d.DTT：1-10mM
e.TritonX-100，Tween20，NP-40，Igepal：0.05％-5％
f. And (3) protease K: 0.1-50 mu g/mL
(3) Reverse transcription of exosome RNAs
The RT-For-All reverse transcription system is (20. mu.L system): 4 μ L of 5 XExoCt RT buffer (i.e., RT-For-All reverse transcription buffer), 0.5 μ LRtase Mix (GeneCopoeia, Inc, USA), and 0.5 μ L LPolyA polymerase (GeneCopoeia, Inc, USA), 1-10 μ L of the lysate from step (2), ddH was added2O is complemented to 20 mu L; mixing, and heating at 37 deg.C for 30min and 85 deg.C for 5 min; after the reaction is finished, ddH can be added according to the abundance of target RNA or DNA to be detected in different samples2Diluting with O0-10 times.
Wherein the RT-For-All reverse transcription buffer solution comprises the following components:
Tris-Cl pH buffer salts: 10-100mM
KCl or NaCl: 10-500mM
c.MgCl2：1-20mM
d.ATP：0.1-10mM
e.dNTP：0.1-1mM
f.DTT：1-15mM
g.miRNA Adapter(GeneCopoeia,Inc，USA)：0.2-2μM
h. Random primers (Thermo Fisher): 0.2-5 mu M
Description of the drawings: the Rtase Mix, PolyA polymerase, and miRNA Adapter are prepripatary series products from GeneCopoeia, Inc, usa, and similar products from other companies may also be used in the reaction.
(4) qPCR detection
qPCR assays can be performed using conventional qPCR assay kits (e.g., available from GeneCopoeia, Inc), and specific procedures can be found in the kit instructions. For example, the qPCR system is (20 μ L system): 10. mu.L of 2 XSSYBR PCR mix (GeneCopoeia, Inc, USA), 0.25. mu.L of universal primers and other specific primers, 2-5. mu.L of the reverse transcription product from step (3), and the addition of ddH2And supplementing O to 20 mu L, and carrying out qPCR detection after uniformly mixing.
The advantages of the invention are as follows:
compared with the traditional method, the detection method provided by the invention has the following advantages:
compared with the traditional RNAzol and other methods for extracting and purifying RNA and the conventional Reverse Transcription (RT) reaction of miRNA and mRNA, the detection method provided by the invention is quick, simple and convenient, and does not need to extract and purify RNA and DNA. In addition, All miRNA and mRNA and other types of RNA (namely RT-For-All) can be subjected to reverse transcription in the same reaction, the Ct value obtained by the detection method provided by the invention is better, and the detection efficiency and the detection sensitivity can be effectively improved.
In particular, the detection of the invention has two innovative points: one is to cleave exosomes directly without extracting RNA, and the other is to reverse transcribe all RNAs in the same reaction for detection.
In the conventional technology, RNA is extracted from exosome, and then different RNA is reverse transcribed by different methods for detection. Generally, the amount of exosomes purified from a sample is very limited, and the RNA content in exosomes is very low, and these trace RNAs are easily lost during extraction and purification, especially small-molecule mirnas with a large proportion of exosomes, which may cause great variation in detection results. The method of the invention releases the content components by directly dissolving the exosome without extracting RNA and DNA, avoids the loss of nucleic acid molecules and has simple and rapid operation.
In the key step of reverse transcription for detecting RNA, the conventional technology is to perform reverse transcription on a large molecule RNA (such as mRNA) and a small molecule RNA (such as miRNA) in different reverse transcription reactions respectively. The conventional mRNA reverse transcription reaction can only detect some macromolecular RNAs such as mRNA, LncRNA and the like, but cannot detect small molecular RNAs such as microRNA and piRNA; the conventional miRNA reverse transcription reaction is only suitable for detecting a few small-molecule RNAs such as microRNA and piRNA, but is not suitable for or incapable of detecting long-molecule RNAs such as mRNA, LncRNA and the like. The reason is that the enzymes and primers used in these two reactions are different and are likely to compete with each other and inhibit each other in one reaction. For example, if the concentration of random primers used in conventional mRNA reverse transcription reaction is too high, it will complementarily bind to the miRNA, piRNA, etc. to be examined, and oiigodt used in conventional mRNA reverse transcription reaction will also compete with miRNA Adapter, thereby inhibiting reverse transcription of miRNA; the invention realizes the simultaneous and high-efficiency reverse transcription of all RNAs by adjusting and optimizing the concentration of random primers used for reverse transcription of mRNA and universal linkers used for reverse transcription of miRNA and removing Oiligoodt used for reverse transcription of conventional mRNA in a reverse transcription reaction system. In addition, the reaction conditions of the reverse transcriptase and the PolyAPolymerase are also different, so that the reaction conditions are further optimized, and the activities of the reverse transcriptase and the PolyAPolymerase are maximized.
In conclusion, the invention realizes the high-efficiency reverse transcription of all RNAs in the same reaction at the same time by the optimal combination of the proportion of the primers and the enzyme and the reaction conditions. On one hand, the method saves the using amount of a sample to be detected, and simultaneously saves time and workload, so that the method can be used for stably and efficiently detecting trace exosome nucleic acid molecules.
The key point of the invention is the design and optimization of the buffer solution, the ExoCt lysis buffer solution can effectively lyse exosomes, protect RNA and DNA released by lysis from degradation by nuclease, and the components of the ExoCt lysis buffer solution do not influence subsequent reverse transcription and PCR reaction. This is an improvement of the present invention.
Furthermore, the invention carries out the optimized combination of reagents on the basis of the conventional method, carries out the reverse transcription of All RNAs in one reaction in the RT reaction (RT-For-All), and overcomes the mutual inhibition effect by designing and optimizing the content of primers in a reverse transcription reaction solution. This is another improvement of the present invention.
Drawings
FIG. 1 shows Ct values for qPCR detection of various RNAs in the experimental group (i.e., Exo-Ct) and the control group in example 1; wherein the amount of exosomes corresponding to the experimental group and the control group in the qPCR reaction is 0.32 mu L of exosome suspension; wherein Exo-Ct refers to the detection result of the experimental group A in example 1; RNA (purified) refers to the results of the detection of experimental group B of example 1.
FIG. 2 shows Ct values for qPCR detection of various RNAs in experimental and control groups in example 2; wherein RT-For-All (lysate) means that the exosome direct lysate of step (2) A in example 2 is subjected to RT-For-All; RT-For-All (RNA) refers to the RNA of the exosome extracted in the step (2) B of the embodiment 2 is subjected to RT-For-All; miRNA RT (RNA) refers to the conventional miRNA reverse transcription reaction of the extracted exosome RNA in the step (2) B in the example 2; mRNA RT (RNA) refers to the conventional mRNA reverse transcription reaction of the extracted exosome RNA of step (2) B of example 2.
FIG. 3 shows Ct values obtained by the detection using the reaction systems in examples 3 to 6.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
Example 1: simple and rapid detection of exosome RNA in Hek293 cell culture solution by one-step method
In this example, the recovery efficiency of RNA from exosome samples extracted using different methods was mainly compared. The exosome direct lysis (one-step) method of the present invention (i.e., a, as experimental group) was compared with the conventional RNA extraction method (multi-step) method (i.e., B, as control group). For comparison, the experimental group and the control group have the same operation steps except that the extraction steps of the exosome RNA sample are different (direct lysis method and conventional RNA extraction method, respectively), and For example, the RT-For-All reverse transcription reaction of the invention is used For detecting exosome nucleic acid in both the experimental group and the control group. The specific operation is as follows:
1. experimental Material
(1) The main apparatus comprises: centrifuge (Thermo), PCR instrument (Takara), real-time fluorescent quantitative PCR instrument (Roche).
(2) The main reagents are as follows: RNAzol RT reagent (GeneCopoeia, Inc, QP020), ExoPure (GeneCopoeia, Inc, USA), ExoCt lysis buffer, ExoCtRT buffer, Rtase Mix (GeneCopoeia, Inc, USA), PolyA polymerase (GeneCopoeia, Inc, USA).
2. Experimental procedure
(1) Exosome extraction
Collecting 5mLHek293(
CRL-1573) adding the cell culture supernatant into a centrifuge tube, centrifuging at 500 Xg for 10min to remove cell debris, transferring the supernatant into a new centrifuge tube, and filtering the cell supernatant with a 0.22 mu MPES filter membrane; then, 1.66mL of ExoSeure (volume ratio of cell culture supernatant to ExoSeure is 3:1) (GeneCopoeia, Inc, USA) was added to the cell culture supernatant, mixed well and then placed at 4 ℃ overnight; the sample was then centrifuged at 6,000rpm for 15min, and the supernatant carefully aspirated, taking care not to touch the exosome pellet; the exosome suspension was resuspended in 50. mu.L of 1 XPBS buffer.
(2) Extraction of exosome nucleic acid samples
Experimental group a direct lysis one-step method using exosomes (inventive method) was as follows:
and (2) taking 5 mu L of the exosome suspension obtained in the step (1), adding 5 mu L of LExoCt lysis buffer solution, uniformly mixing, placing on ice for 5min, then placing at 75 ℃ for 10min, and placing on ice after the reaction is finished.
The composition of the ExoCt lysis buffer is as follows:
Tris-Cl pH buffer salts: 50mM
b.BSA：10μg/mL
RNase inhibitor: 2U/. mu.L
d.TritonX-100，Tween20，NP-40，Igepal：0.5％
e. And (3) protease K: 10. mu.g/mL.
Control group B uses RNAzol to extract exosome RNA (conventional method), as follows:
taking 40 μ L of the exosome suspension obtained in step (1), adding 100 μ L of LRNAzol RT reagent (GeneCopoeia, Inc, QP020), extracting RNA according to the conventional RNAzol operation manual, and finally extracting with 80 μ L of ddH2O lysis of RNA.
(3) RT-For-All reverse transcription of exosome RNA
The reverse transcription system was (20 μ L system): 4 μ L of 5 XExoCtRT buffer, 0.5 μ LRtase Mix (GeneCopoeia, Inc, USA) (where,reverse transcriptase content 100U), and 0.5. mu.L of LPolyA polymerase (GeneCopoeia, Inc, USA) (wherein PolyA polymerase content is 2U), 4. mu.L of lysate (A) from experimental group or 4. mu.L of RNA (B) from control group RNAzol purification, ddH was added2O is complemented to 20 mu L; mixing, and heating at 37 deg.C for 30min and 85 deg.C for 5 min; after completion of the reaction, the reverse transcription product was diluted 5-fold to 100. mu.L.
The composition of the ExoCt RT buffer solution is as follows:
Tris-Cl pH buffer salts: 50mM
KCl or NaCl: 100mM
c.MgCl2：5mM
d.ATP：1mM
e.dNTP：0.2mM
f.DTT：5mM
g.miRNA Adapter(GeneCopoeia,Inc，USA)：0.5μM
h. Random primers (Thermo Fisher): 2 μ M.
(4) qPCR detection
qPCR assays can be performed using conventional qPCR assay kits (e.g., available from GeneCopoeia, Inc), and specific procedures can be found in the kit instructions. For example, the qPCR system is (20 μ L system): mu.L of 2 XSSYBR PCR mix (GeneCopoeia, Inc, USA), 0.25. mu.L of universal primers and other specific primers (all available from GeneCopoeia, Inc, USA), 2. mu.L of 5-fold diluted reverse transcription product was added and mixed well for qPCR detection. Description of the drawings: the universal primers and other specific primers used in the experiments were prepracticary series products from GeneCopoeia, Inc, usa, and similar primer products from other companies can also be used in the reactions.
3. Results of the experiment
The real-time fluorescent quantitative PCR instrument carries out qPCR to detect a plurality of RNAs, and the Ct obtained is shown in figure 1. As can be seen from fig. 1: under the condition that the corresponding exosome amount in the qPCR reaction is the same, compared with the traditional method for extracting exosome RNA, the method for extracting exosome RNA has the advantages that the Ct value of the RNA obtained by the method is better, the loss of the RNA is less, and the detection efficiency and the detection sensitivity can be effectively improved.
Example 2: simple and rapid detection of exosome RNA in Mesenchymal Stem Cell (MSC) culture solution by one-step method
In this example, the method of the present invention was applied to exosome detection of Mesenchymal Stem Cells (MSCs) in its entirety. According to the latest literature report, miRNA and housekeeping RNA with markers in Mesenchymal Stem Cell (MSC) exosomes are selected for detection, and the miRNA and housekeeping RNA specifically comprise Mir23a-3p, Mir125b, Mir1246a and the like.
In this example, the RT-For-All reverse transcription reaction in the method of the present invention (i.e., a, experimental group) was compared with the conventional miRNA reverse transcription reaction (i.e., B, control 1) and the conventional mRNA reverse transcription reaction (i.e., C, control 2) in the reverse transcription step of the exosome RNA detection method.
Among them, the conventional reverse transcription reaction of mRNA (i.e., control 2) can detect only some large RNA such as mRNA, LncRNA, etc., but cannot detect small RNA such as microRNA and piRNA; whereas the conventional miRNA reverse transcription reaction (i.e., control 1) is suitable for detecting only some small RNAs, such as microRNA and piRNA, and is not suitable or capable of detecting long RNAs, such as mRNA, LncRNA, etc. The RT-For-All reverse transcription reaction of the invention can simultaneously and efficiently reverse transcribe All RNAs in the same reaction.
1. Experimental Material
(1) The main apparatus comprises: centrifuge (Thermo), PCR instrument (Takara), real-time fluorescent quantitative PCR instrument (Roche).
(2) The main reagents are as follows: RNAzol RT reagent (GeneCopoeia, Inc, QP020), ExoPure (GeneCopoeia, Inc, USA), ExoCt lysis buffer, ExoCtRT buffer, Rtase Mix (GeneCopoeia, Inc, USA), PolyA polymerase (GeneCopoeia, Inc, USA).
2. Experimental procedure
(1) Exosome extraction
Collect 5mL mesenchymal stem cells (MSC, (R))
ACS-7010)), adding the supernatant to a centrifuge tube, centrifuging at 500 × g for 10min to remove cell debris, transferring the supernatant to a new centrifuge tube, and filtering the cell supernatant with a 0.22 μ M PES filter membrane; in-line with the aboveAdding 1.66mL of ExoSere (GeneCopoeia, Inc, USA) (the volume ratio of the cell culture supernatant to the ExoSere is 3:1) into the cell culture supernatant, mixing well, and standing at 4 ℃ overnight; the sample was then centrifuged at 6,000rpm for 15min, and the supernatant carefully aspirated, taking care not to touch the exosome pellet; the exosome suspension was resuspended in 50. mu.L PBS buffer.
(2) Exosome nucleic acid sample extraction
A. Direct lysis one-step method of exosomes (method of this patent):
and (2) taking 5 mu L of the exosome suspension obtained in the step (1), adding 5 mu L of LExoCt lysine buffer, uniformly mixing, placing on ice for 5min, then placing at 75 ℃ for 10min, and placing on ice after the reaction is finished.
RNAzol extraction of exosome RNA (conventional method)
Taking 40 μ L of the exosome suspension obtained in the step (1), adding 100 μ L of LRNAzol RT reagent, and extracting RNA according to the RNAzol operation manual. Finally using 80. mu.L ddH2O lysis of RNA.
(3) RT reaction
A. This patent RT-For-All reverse transcription reaction: 4 mu.L of the one-step lysate of the exosome obtained in the step (2) A or 4 mu.L of the purified exosome RNA obtained in the step (2) B is subjected to reverse transcription by using an RT-For-All system. The reverse transcription system is shown in example 1.
B. Conventional miRNA reverse transcription reaction: mu.L of the purified exosome RNA obtained in step (2) B was reverse transcribed using a conventional miDNA synthesis system (purchased from GCI).
C. Conventional mRNA reverse transcription reaction: mu.L of the purified exosome RNA obtained in step (2) B was reverse transcribed using a conventional cDNA synthesis system (purchased from GCI).
(4) qPCR reaction: the reverse transcription products were diluted 5-fold, and 2. mu.L each was taken for qPCR detection.
3. Results of the experiment
The real-time fluorescent quantitative PCR instrument performs qPCR to detect a plurality of RNAs, and the obtained Ct is shown in figure 2. As can be seen from the results of fig. 2: the RT-For-All reverse transcription reaction in the invention is the high-efficiency combination of two traditional RT methods, and the RT-For-All reverse transcription reaction is combined with the direct cracking one-step method of exosome, thereby not only greatly improving the detection time efficiency, but also further improving the detection sensitivity. Even For purified RNA, RT-For-All reverse transcription reactions have the same or better sensitivity than the two traditional RT methods.
Example 3:
the procedure of example 3 was similar to that of the experimental group of example 1, and the sample was an ExoCt exosome lysate obtained by lysing exosomes with an ExoCt lysis buffer, except that the concentrations of reverse transcriptase, PolyA polymerase, miRNA Adapter and random primer in the reverse transcription reaction system were different, as shown in table 1. In the reverse transcription reaction system, the concentration of reverse transcriptase is 1U/. mu.L of the reaction system, and the concentration of PolyA polymerase is 0.05U/. mu.L of the reaction system; the concentration of miRNA Adapter in RT buffer was 0.2. mu.M and the concentration of random primers was 0.2. mu.M in the reverse transcription reaction. The detection results are shown in fig. 3, and are found to achieve similar technical effects.
TABLE 1 examples 3-6 reverse transcription reaction System component concentrations
| RT-For-ALL | Example 3 | Example 4 | Example 5 | Example 6 |
| Reverse transcriptase (U/. mu.L) | 1 | 10 | 4 | 7 |
| PolyA polymerase (U/. mu.L) | 0.05 | 0.5 | 0.1 | 0.3 |
| miRNA Adapter(μM) | 0.2 | 2 | 0.8 | 1.5 |
| Random primer (mu M) | 0.2 | 5 | 2 | 3 |
Example 4:
the procedure of example 4 was similar to that of the experimental group of example 1, and the sample was ExoCT exosome lysate, except that the concentrations of reverse transcriptase, PolyA polymerase, miRNA Adapter and random primer in the reverse transcription reaction system were different, as shown in table 1. In the reverse transcription reaction system, the concentration of reverse transcriptase is 10U/. mu.L of the reaction system, and the concentration of PolyA polymerase is 0.5U/. mu.L of the reaction system; the concentration of miRNA Adapter in RT buffer was 2. mu.M and the concentration of random primers was 5. mu.M in the reverse transcription reaction. The detection results are shown in fig. 3, and it is found that similar technical effects can be achieved.
Example 5:
the procedure of example 5 was similar to the experimental group of example 1, and the sample was ExoCT exosome lysate, except that the concentrations of reverse transcriptase, PolyA polymerase, miRNA Adapter and random primer in the reverse transcription reaction system were different, as shown in table 1. In the reverse transcription reaction system, the concentration of reverse transcriptase is 4U/. mu.L of the reaction system, and the concentration of PolyA polymerase is 0.1U/. mu.L of the reaction system; the concentration of miRNA Adapter in RT buffer was 0.8. mu.M and the concentration of random primers was 2. mu.M in the reverse transcription reaction. The detection results are shown in fig. 3, and it is found that similar technical effects can be achieved.
Example 6:
the procedure of example 6 was similar to that of the experimental group of example 1, and the sample was ExoCT exosome lysate, except that the concentrations of reverse transcriptase, PolyA polymerase, miRNA Adapter and random primer in the reverse transcription reaction system were different, as shown in table 1. In the reverse transcription reaction system, the concentration of reverse transcriptase is 7U/. mu.L of the reaction system, and the concentration of PolyA polymerase is 0.3U/. mu.L of the reaction system; the concentration of miRNA Adapter in RT buffer was 1.5. mu.M and the concentration of random primers was 3. mu.M in the reverse transcription reaction. The detection results are shown in fig. 3, and it is found that similar technical effects can be achieved.
In conclusion, the Ct value of the exosome direct lysis one-step method is better than that of the traditional RNAzol extraction method (see example 1 and figure 1), which shows that the loss of RNA is less, the method is simple and convenient, and the detection efficiency and the detection sensitivity can be effectively improved. Meanwhile, the RT-For-All reverse transcription reaction in the invention is the high-efficiency combination of two traditional RT methods (namely miRNA reverse transcription reaction and mRNA reverse transcription reaction), can simultaneously detect macromolecular RNA and small molecular RNA, and combines the macromolecular RNA and the small molecular RNA with a direct cracking one-step method of exosome, thereby not only greatly improving the detection time, but also further improving the detection sensitivity (see example 2 and figure 2).
As can be seen from examples 3-6 (FIG. 3), the RT-For-All reverse transcription reaction of the present invention has several main components within a certain concentration range, such as the reverse transcriptase concentration of 1-10U/. mu.L reaction system, and the PolyA polymerase concentration of 0.05-0.5U/. mu.L reaction system; the concentration of miRNA Adapter in RT buffer solution is 0.2-2 μ M, and the concentration of random primer is 0.2-5 μ M, both can work well. For samples from different sources and different detection genes, the optimal effect can be achieved by properly optimizing the mixture ratio in the range (as in example 5).
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting nucleic acid molecules in exosomes, comprising the steps of:
lysing exosomes in the sample for releasing exosome nucleic acid molecules; preferably, the sample is a cell culture fluid, plasma, urine or other bodily fluid sample;
performing reverse transcription reaction, wherein the exosome is subjected to reverse transcription reaction, namely RT-For-All, without extracting nucleic acid molecules or after extracting nucleic acid molecules after cracking, so that All RNAs in the exosome are subjected to reverse transcription in the same reaction;
wherein a reverse transcription buffer comprising a universal adaptor (e.g., miRNA Adapter) and a random primer is used in the reverse transcription reaction;
preferably, the reverse transcription reaction is followed by quantitative PCR or sequencing detection.
2. The detection method of claim 1, wherein the nucleic acid molecule comprises RNA (e.g., mRNA, miRNA, lncRNA, cirRNA) and DNA.
3. The detection method according to claim 1, wherein in the step of lysing the exosomes, lysis is performed using an exosome lysis buffer, wherein the lysis buffer comprises a pH buffer salt (preferably Tris-Cl), BSA, an RNase inhibitor and a non-ionic detergent;
preferably, the non-ionic detergent is TritonX-100, Tween20, NP-40, Igepal in any one or more combinations.
4. The assay of claim 1, wherein the lysis buffer further comprises proteinase K for inhibiting nuclease activity and protecting nucleic acids from degradation; preferably, the lysis buffer further comprises DTT.
5. The detection method according to claim 1, wherein a reverse transcription reaction system comprising a reverse transcription buffer, a reverse transcriptase, and PolyA polymerase, preferably further comprising dNTP and ATP, is used in the reverse transcription reaction step.
6. The detection method according to claim 1, wherein the conditions of the reverse transcription reaction include 37 ℃ to 42 ℃ for 5 to 30 min.
7. The detection method according to claim 1, wherein the product of RT-For-All reverse transcription reaction can be directly used as a substrate For various detections, such as nucleic acid detection For quantitative PCR (qPCR); can also be used as a library construct for nucleic acid NGS.
8. A kit for detecting nucleic acid molecules in exosomes, comprising a reverse transcription reaction system; wherein the reverse transcription reaction system comprises a reverse transcription buffer solution, a reverse transcriptase and a PolyA polymerase, and preferably also comprises dNTP and ATP;
wherein the reverse transcription buffer comprises a universal linker (e.g., miRNA Adapter) for reverse transcription of small RNA molecules and a random primer for reverse transcription of large RNA molecules.
9. The kit of claim 8, further comprising a lysis buffer, wherein the lysis buffer comprises pH buffering salts (preferably Tris-Cl), BSA, RNase inhibitors, and a non-ionic detergent;
preferably, the non-ionic detergent is TritonX-100, Tween20, NP-40, Igepal in any one or more combinations.
10. The kit of claim 9, wherein the lysis buffer further comprises proteinase K for inhibiting nuclease activity and protecting nucleic acids from degradation; preferably, the lysis buffer further comprises DTT.
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