Co-extraction method and application of mRNA and protein in trace sample

Abstract

The invention belongs to the technical field of biological analysis, and particularly relates to a co-extraction method for mRNA and protein in a trace sample and application. The co-extraction method mainly comprises the following steps: capturing messenger RNA by adopting Oligo dT magnetic beads so as to separate the messenger RNA from protein, and then selecting a proteome pretreatment working process based on SP3 magnetic beads. According to the technology, Oligo dT magnetic beads are innovatively selected to separate messenger RNA. The surface of the Oligo dT magnetic bead is modified with an Oligo dT sequence, so that the Oligo dT magnetic bead can selectively capture mRNA. The mRNA separation and purification technology has the characteristics of high efficiency, high purity and simplicity and convenience in operation. The problems that mRNA and protein in a trace sample are difficult to separate, the RNA amount is small, follow-up analysis cannot be met, and the proteomics pretreatment process is low in efficiency can be solved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological analysis, and in particular relates to a method for co-extracting mRNA and protein in a trace sample and an application thereof. Background Art

[0002] In the past decade, spatial transcriptomics has made a major leap from visualizing a few genes to being able to detect the entire transcriptome at the same time, greatly promoting new discoveries in different fields such as neuroscience, immunity and development. According to the central dogma, transcriptome information must be translated into proteins in order to exert the functional effects of genes. Furthermore, the expression levels of mRNA and protein in all tissues and cells are not linearly correlated, and the determination of transcriptional profiles cannot replace the characteristic information of the proteome. Therefore, the study of spatial proteomics is very important. Traditional methods such as transcriptomics and proteomics can usually only provide information at a specific molecular level and cannot show their distribution in tissue structures. Spatial multi-omics technology fills this gap. Therefore, the combined analysis of spatial proteomics and spatial transcriptomics can better and more accurately explain the spatial expression and distribution patterns of molecules such as genes and proteins, better understand their functions and relationships, and help reveal new biological mechanisms, signaling pathways and regulatory networks.

[0003] Currently, the technology that allows the joint determination of spatial transcriptome and spatial proteome is still limited. Usually, the transcriptome and proteome are studied on two adjacent slices, and then the data are integrated and analyzed. However, there will be sample heterogeneity in different slices, and even adjacent parts will differ in structure and cell composition. Therefore, methods for measuring protein markers and transcriptomes in the same tissue slice appeared later. Such as Nanostring's GeoMxDSP technology and Ben-Chetrit, N et al.'s SPOTS (Spatial Protein and Transcriptome Sequencing) technology, this type of technology can simultaneously analyze transcriptomes and multiple proteins. However, this type of technology can only study known proteins, and the number of proteins that can be analyzed simultaneously is limited. Therefore, there is an urgent need for a technology that can analyze proteins broadly at the spatial level while also obtaining transcriptome data.

[0004] Laser capture microdissection (LCM) is a cutting-edge method for acquiring spatial information. It can quickly collect regions of interest (ROIs) from heterogeneous tissues with a resolution ranging from single cells to cell groups. Therefore, LCM technology has been widely used in spatial multi-omics research. However, there are currently few methods that use laser microdissection technology to selectively collect ROIs on pathological sections and simultaneously extract RNA and proteins for multi-omics analysis. The main reason is that the RNA and protein content in trace samples is small and difficult to separate, making it difficult to obtain enough protein and RNA for subsequent multi-omics analysis. Therefore, here we propose a new solution. Summary of the invention

[0005] At present, it is difficult to separate RNA and protein in micro-samples cut by laser microdissection, and the amount of RNA is small and cannot meet the needs of subsequent transcriptome sequencing analysis and proteomics pre-processing. The present invention provides a method for co-extraction of mRNA and protein in the same micro-sample, which can simultaneously obtain mRNA and protein in the sample. The application of this technology can be carried out in a volume as small as 0.01mm 3 mRNA and protein were extracted simultaneously from mouse brain tissue weighing approximately 0.0103 mg. Compared with the traditional Trizol method, the starting amount of sample was greatly reduced.

[0006] The main technical solutions of the present invention are as follows:

[0007] In a first aspect, the present application provides a method for co-extracting mRNA and protein from a trace sample, comprising the following steps: using Oligo dT magnetic beads to capture messenger RNA to separate it from protein, and then using SP3 magnetic beads to purify the protein.

[0008] According to the specific implementation of this application, the specific method includes:

[0009] (1) Place tissue block slices on the laser microdissection stage, use laser microdissection technology to collect areas of interest on the tissue slices, and obtain tissues of the same size;

[0010] (2) placing the tissue from step (1) into a lysis solution for lysis;

[0011] (3) After the lysis is completed, the mRNA is extracted and separated by magnetic bead separation, and reverse transcribed into cDNA and analyzed by real-time fluorescence quantitative PCR;

[0012] (4) reducing and alkylating the protein in the lysate after extracting mRNA in step (3); purifying;

[0013] (5) The protein purified in step (4) is dissolved in a mass spectrometry-compatible lysis buffer and subjected to enzymatic hydrolysis.

[0014] (6) Add formic acid solution to step (5) to acidify and terminate the reaction, freeze-dry, and re-dissolve to prepare a peptide solution.

[0015] According to a specific embodiment of the present application, the lysis buffer in step (2) is selected from at least one of RLTplus lysis buffer, RIPA lysis buffer and Tris-HCl lysis buffer containing 1% SDS.

[0016] According to a specific implementation of the present application, the cDNA in step (3) can also be prepared using the smart-seq2 library construction method and transcriptome analysis using the second-generation sequencing technology.

[0017] According to a specific embodiment of the present application, the purification method in step (4) is selected from at least one of ultrafiltration membrane purification method (i.e., FASP method), SP3 magnetic bead purification method and organic solvent precipitation method.

[0018] According to a specific embodiment of the present application, in step (5), the protein purified in step (4) is dissolved in a mass spectrometry-compatible lysis buffer, such as a 25 mM ammonium bicarbonate solution, and Trpsin enzyme is added for enzymatic hydrolysis.

[0019] According to a specific implementation of the present application, in step (5), formic acid solution is added to the enzymatic hydrolysis system to acidify and terminate the reaction, the reaction is freeze-dried, and the peptide solution is prepared by re-dissolving with 0.1 wt % formic acid solution.

[0020] Ammonium bicarbonate solution is used as a mass spectrometry-compatible lysis solution, and residual ammonium bicarbonate can enter the mass spectrometer without affecting the detection; adding formic acid to acidify plays a role in adjusting the pH of the solution, and the pH of the sample entering the mass spectrometer analysis needs to be around 3.

[0021] According to a specific embodiment of the present application, the trace peptide sample in step (6) is subjected to liquid chromatography-mass spectrometry analysis and a whole proteomics quantitative analysis.

[0022] The present invention provides a stable and efficient protein and mRNA co-extraction method for trace samples, which is not only applicable to frozen embedded samples but also to cell suspensions, etc. The method innovatively selects Oligo dT magnetic beads to separate messenger RNA. The surface of Oligo dT magnetic beads is modified with Oligo dT sequences, i.e., oligonucleotides containing multiple thymines (T). These Oligo dT sequences can complementarily pair with the PolyA tail structure at the 3' end of the eukaryotic mRNA molecule to combine the mRNA with the magnetic beads. This mRNA separation and purification technology has the characteristics of high efficiency, high purity and simple operation.

[0023] In addition, the present invention also uses a proteomic pretreatment workflow based on paramagnetic particles, driving protein enrichment on the surface of carboxyl or other hydrophilic magnetic beads through organic solvents such as ethanol or acetonitrile. Using this mechanism, the SP3 magnetic bead method can achieve unbiased recovery of proteins and peptides, is compatible with a large number of compounds (such as detergents, chaotropes and salts), and promotes bottom-up deep proteomic analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention provides a co-extraction workflow of mRNA and protein in a micro sample by laser microdissection;

[0025] Figure 2 Comparison of RNA quality of samples prepared under different lysis conditions in the present invention;

[0026] Figure 3 It is a comparison of the number of protein identifications of samples prepared under different lysis conditions in the present invention;

[0027] Figure 4 Overlap graph of proteins identified under different cleavage conditions in the present invention;

[0028] Figure 5 Comparison of protein identification numbers of different protein purification methods in the present invention;

[0029] Figure 6 Overlap diagram of proteins identified by different protein purification methods in the present invention. DETAILED DESCRIPTION

[0030] Embodiments of the present invention will be described in detail below in conjunction with examples. However, the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Except for the reagents and instruments noted in the examples, the types, models, qualities, properties or functions of the remaining reagents and instruments used, the same or similar materials can all be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0031] The main instruments used in the following specific examples are shown in Table 1

[0032] Table 1. Main instrument names

[0033]

[0034] The main reagents / consumables used in the following specific examples are shown in Table 2:

[0035] Table 2. Materials and reagents

[0036]

[0037]

[0038] The present invention is suitable for fresh tissue samples, frozen embedded tissue samples and cell suspensions of various animals, and can also be widely used in clinical samples. The specific implementation case is OCT-embedded mouse brain tissue stored in a -80°C refrigerator for 3 months. The specific experimental steps are as follows: Figure 1 shown.

[0039] Example 1 Embedding, Slicing and Laser Microdissection of Mouse Brain Tissue

[0040] 1.1 Sample preparation

[0041] Preparation: Pour 1 / 3 of OCT into the embedding box and place the embedding box on ice to pre-cool. Place the entire tube of OCT on ice to pre-cool.

[0042] The mice were deeply anesthetized with 1% sodium pentobarbital and perfused transcardially with 1× phosphate buffered saline (PBS). The intact brain of the mouse was carefully removed using surgical scissors and surgical forceps. The blood around the mouse brain tissue was washed with 1× PBS, and the surface liquid was wiped dry with dust-free paper. The tissue was transferred to a pre-cooled embedding box with a spatula and the direction of the section was adjusted. Pre-cooled OCT was added to the embedding box, and the tissue was gently touched with a spatula to make the tissue fit completely with the OCT. Mark the position, place it on dry ice to completely solidify it, and then store it in a -80° refrigerator.

[0043] 1.2 Slicing

[0044] Clean the cryostat, insert a new sterile blade, set the cryostat of the cryostat to -20 °C, set the cutting thickness to 10 μm, remove the frozen block of OCT compound from the cryo-mold, trim if necessary, and stick it on the metal stage of OCT for cryosectioning. Section the tissue and stick it on PET film slides.

[0045] The prepared tissue sections were fixed in cold 75% (vol / vol) ethanol for 10 seconds. This step can wash away the excess OCT, and then the tissue sections were dehydrated in 90% and 100% (vol / vol) ethanol for 10 seconds. The slides were placed at room temperature for 1-2 minutes to dry.

[0046] 1.3 Laser microcapture cutting

[0047] Place the mouse brain tissue slices on the stage of the laser microdissection instrument, and find the cortex and ventricle area of ​​the mouse brain through the microscope of the instrument. Select the By pass function on the laser microdissection instrument and set the cutting area to 10 mm 2Afterwards, the tissue was cut and sampled, and the selected area was collected into a 0.2 ml PCR tube. The volume of tissue cut in this step was calculated to be 0.1 mm 3 , the tissue mass can be estimated to be approximately 0.103 mg.

[0048] 2. Tissue Lysis and mRNA Isolation

[0049] 2.1 Add 30 μl of Buffer RLT Plus for lysis, pipette and beat repeatedly to ensure complete lysis, centrifuge the PCR tube containing tissue and lysis solution at 5000g for 1 minute, and then store it in a -80℃ refrigerator and take it out when needed.

[0050] 2.2 Place the Oligo dT magnetic bead bottle on a vortex oscillator for 20 seconds and vortex to fully resuspend the magnetic beads. Use a pipette to transfer the required volume of magnetic beads to a new centrifuge tube. Add the same volume of binding buffer and resuspend the magnetic beads. Place the centrifuge tube on a magnetic rack, let it stand for 1 minute, and discard the supernatant. Add the same volume of binding buffer as the initial volume and set aside.

[0051] 2.3 Add 10 μl of the prepared magnetic beads to the lysate described in 2.1, shake thoroughly to mix, and incubate at room temperature for 5 minutes. Note: If the solution is viscous, increase the binding time.

[0052] 2.4 Place the PCR tube on the magnetic stand for 1-2 minutes, and pipette the supernatant containing protein into a new PCR tube for subsequent experiments. Oligo dT magnetic beads bound to mRNA need to be washed twice: once with 60μl washing buffer ①, and once with 60μl washing buffer ②. Note: The magnetic beads need to be thoroughly resuspended during washing.

[0053] 2.5 Then add 11 μl enzyme-free water or TE buffer. Incubate at 65℃-75℃ for 2 min, then place the centrifuge tube on a magnetic rack and quickly transfer 10 μl of the supernatant containing mRNA to a new RNase-free centrifuge tube.

[0054] Example 3 Detection of mRNA extracted from mouse brain

[0055] 3.1 Reverse transcription

[0056] (1) Prepare the reverse transcription reaction solution according to the following recipe and mix well for later use.

[0057] Table 3. Ratio of each group in the reverse transcription reaction solution

[0058]

[0059]

[0060] Add 10 μl of the prepared reverse transcription reaction solution to the mRNA prepared in the previous step. Perform the following procedure on the PCR instrument:

[0061] Table 4. Reverse transcription procedures

[0062]

[0063] (2) Prepare PCR reaction solution according to the following formula

[0064] Table 5: Distribution ratio of each group of PCR reaction solution

[0065]

[0066] Perform the following program on a thermal cycler:

[0067] Table 6. PCR cycle program

[0068]

[0069] The number of PCR cycles depends on the amount of RNA input. Generally, 16-19 cycles are suitable for most applications.

[0070] (3) PCR product purification

[0071] According to the instructions of the PCR product purification kit, add 1.8 times the magnetic beads to the PCR product, blow to mix, incubate at room temperature for 5 minutes, let it stand on the magnetic rack, and discard the supernatant overnight. Wash the magnetic beads with freshly prepared 80% ethanol, let it stand on the magnetic rack, discard the supernatant overnight, repeat this step once, and then dry the magnetic beads at room temperature. Note: There is no need to resuspend the magnetic beads thoroughly. Be careful not to crack the magnetic beads when drying them, about 2 minutes. Resuspend the magnetic beads thoroughly with 30μl of enzyme-free water or TE buffer, incubate at room temperature for two minutes, and elute the cDNA from the magnetic beads.

[0072] (4) cDNA concentration detection

[0073] The concentration of purified cDNA was detected by Qubit dsDNAHS detection kit on Qubit4.0. The cDNA fragment distribution was detected on Agilent 4200. The cDNA peak should be around 1-2kb. Note: cDNA should be stored in a -80℃ refrigerator for a long time. After this step, cDNA can be directly used for subsequent DNA library construction or fluorescence quantitative PCR analysis.

[0074] 3.2 Fluorescence quantitative PCR analysis

[0075] Take 2 μl of purified cDNA and perform 20 μl qPCR reaction according to the instructions of the qPCR kit. The mRNA extraction efficiency can be evaluated by detecting the expression of housekeeping genes such as GAPDH and ACTB.

[0076] 4. Protein Extraction and Peptide Preparation

[0077] 4.1 Protein reductive alkylation

[0078] Add 100 mM DTT solution to the protein-containing lysate reserved in step 2.4 to a final concentration of about 10 mM. React at 37°C for 30 minutes and then spin off instantly. Add IAM solution with a final concentration of 55 mM under light-proof conditions and react in a dark room for 45 minutes.

[0079] 4.2 Protein purification and enzymatic hydrolysis

[0080] (1) Preparation of SP3 magnetic beads: Sera-Mag Speed ​​Bead carboxyl magnetic beads (50 mg / ml) need to be mixed with hydrophilic and hydrophobic magnetic beads in a ratio of 1:1. Take 100 μL of each type of magnetic beads and mix thoroughly, place on a magnetic stand and let stand for 60 seconds, discard the supernatant, add 1 ml of ultrapure water and mix well, and set aside. Note: The prepared magnetic beads can be stored in a -4°C refrigerator for one month.

[0081] (2) Add 10 μl of the prepared SP3 magnetic beads and gently shake to mix. Add 1% formic acid at a volume ratio of 10:1 to adjust the pH to acidic (about 2); immediately add acetonitrile at a final concentration of 50% (v / v), gently shake to mix; react at room temperature for 8 minutes.

[0082] (3) Place the PCR tube on the magnetic rack for 2 minutes. After the magnetic beads are completely absorbed on the tube wall, gently aspirate and discard the supernatant to avoid contact with the magnetic beads; remove the PCR tube from the magnetic rack, add 200 μl of 70% ethanol, gently shake and mix, place the PCR tube on the magnetic rack for 30 seconds, wait for the magnetic beads to be completely absorbed on the tube wall, gently aspirate and discard the supernatant, and repeat this step; remove the PCR tube from the magnetic rack, add 180 μl of 100% acetonitrile, gently shake and mix, centrifuge, place the PCR tube on the magnetic rack for 15 seconds, wait for the magnetic beads to be completely absorbed on the tube wall, gently aspirate and discard the supernatant, and evaporate naturally for 30 seconds;

[0083] (4) Prepare enzyme solution: Take 27 μl of 25 mM ammonium bicarbonate solution, add 3 μl of 100 ng / μl trypsin, add 30 μl of the prepared enzyme solution to the PCR tube, shake and mix, and incubate at 37°C overnight. Note: The magnetic beads need to be completely resuspended in this step.

[0084] (5) Centrifuge the sample after enzymatic hydrolysis in (4) at 12000 rpm for 3 minutes at room temperature to fully centrifuge the liquid on the tube wall; then place the PCR tube on a magnetic rack and let it stand for 2 minutes, carefully aspirate the supernatant, and the peptide sample is obtained. Note: 5mm 2 The above tissues can be used for peptide quantification.

[0085] (6) Add 1 μl of 10 wt% formic acid solution to acidify the peptide sample, freeze-dry the acidified peptide, and then re-dissolve it in 50 μl of 0.1 wt% formic acid solution.

[0086] 5. Mass spectrometry identification and data processing of peptide fragments.

[0087] Take 1 μl of the peptide solution obtained in step 4.2 for proteomics analysis. Chromatographic separation was performed using nanoElute, a nanoliter liquid chromatograph, and mass spectrometry detection was performed using a trapped ion mobility time-of-flight mass spectrometer timsTOFpro2. The peptides were separated using a self-packed C18 column (75 μm column diameter, 25 cm column length, 1.9 μm column particle size). The peptides were eluted on the nLC system by a gradient elution program, eluting 2%–22% buffer B (0.1% formic acid and acetonitrile) within 0–45 min, 22%–35% buffer B within 45–50 min, 35%–80% buffer B within 50–55 min, and 40%–100% buffer B within 53–65 min. The end of the nanoliter liquid separation was directly connected to the mass spectrometer.

[0088] The peptides separated by liquid phase are ionized by the Captive Spray source and then enter the tandem mass spectrometer timsTOFPro2 for DDA (Data Dependent Acquisition) mode detection.

[0089] Main parameter settings: ion source voltage is set to 1.7 kV; ion mobility range is 0.6-1.6 VS / cm 2 , set the scan mode of the quadrupole to analyze the precursor ions of the mass spectrometer using a 2Th window of m / z < 700 and a 3Th window of m / z > 700 in the total m / z range of 100–1.700. The collision energy decreases linearly with increasing mobility, from 1 / K0 = 1.6 Vscm -2 59eV to 1 / K0=0.6Vscm -2The MS / MS precursor was scanned again at an intensity threshold of 500 arbitrary units (au) until a “target value” of 20.000 au was reached, with a dynamic exclusion time set to 40 s. For data-independent analysis, we exploited the correlation of ion mobility with m / z and synchronized the elution of the precursor in each ion mobility scan with the quadrupole isolation window. The collision energy was varied as a function of ion mobility from 1 / K0 = 1.6 Vscm -2 The linear rise from 59eV to 1 / K0 = 0.6Vscm -2 20eV at the time.

[0090] The raw data obtained using timsTOFPro2 were processed by MaxQuant (2.0.1.0) for peptide annotation against the UniProt database (released in 2022, UP000005640_9606). For peptide searches, the minimum length of identified peptides was set to 7 amino acids; in trypsin digestion, the number of missed cuts allowed was set to 2; N-terminal acetylation and methionine oxidation were set as variable modifications, and cysteine ​​carbamidomethylation was set as a fixed modification; Match betweenruns mode was checked, and FDR < 1% was set to screen the identified peptides and proteins.

[0091] In addition, the mRNA and protein co-extraction method in laser capture microdissection microsamples also underwent comparative tests on the lysis efficiency of different lysis buffers and on different protein separation and purification methods.

[0092] (1) Comparative test of different lysis buffers:

[0093] In step 4 above, we compared three different lysis buffers. The reagents required in this case were all prepared with DECP water, and the consumables used were all nuclease-free consumables. The samples used for comparison were 10 μm thick mouse brain tissue slices, and laser microdissection cut 10 mm 2 of tissues (n=5).

[0094] Lysis buffer 1 is RIPA lysis buffer, which is a traditional rapid lysis buffer that can be used to lyse cells or tissues. The protein obtained after lysis can generally be directly used in conventional WB, IP and other experiments. Its specific composition is: 50mM Tris, 150mM NaCl, 1% TritonX-100, 1% SDC, 0.1% SDS.

[0095] Lysis buffer 2 is a lysis buffer containing 1% SDS. The main formula is 100mM Tris-HCl (pH7.5), 500mM NaCl, 10mM EDTA, 1% SDS, 5mM DTT.

[0096] Lysis buffer 3 is RLT Plus buffer, containing guanidine isothiocyanate, TritonX-100, etc. It is a lysis buffer used for lysing cells and tissues before RNA or RNA / DNA separation, which can effectively inhibit RNA degradation.

[0097] Result analysis:

[0098] The samples used for comparison here are 10 μm thick mouse brain tissue slices, laser microdissection cut 10 mm 2 Tissues (n=5), here in step 2.1, the above three lysis solutions were tried respectively. The extracted RNA was reverse transcribed into cDNA, and the CT value of the GAPDH gene was compared by fluorescence quantitative PCR to determine the efficiency of RNA extraction; and the protein was enzymatically hydrolyzed into peptides, the peptides were frozen and dried, and re-dissolved in 50μl of 0.1wt% formic acid solution, and 1μL was uniformly taken for LC-MS / MS analysis. Note: The amount of peptides loaded here is 1 / 50 of each sample.

[0099] Figure 2 The Ct value of the GAPDH gene after RT-qPCR reaction of mRNA extracted from three lysates. From the results, the Ct value of the GAPDH gene after using Lysis buffer 3 is the smallest, indicating that the extraction efficiency of mRNA in this lysate is the highest; Figure 3 In order to unify the number of protein identifications after mass spectrometry detection using 1 / 50 of the total peptides, the results show that the number of protein identifications using Lysis buffer 3 is the highest. Lysis buffer 3 contains guanidine isothiocyanate, which can inhibit RNase and denature other proteins. To explore whether this lysate has an effect on protein identification, an Overlap graph is made for the proteins identified by the three lysates. Figure 4 As shown, it can be seen that the proteins identified by the three lysates are highly repetitive, so the use of Lysis buffer 3 does not have much effect on the identification of proteins. Therefore, the present invention finally chooses to use this lysate, namely, RLTPlus buffer.

[0100] (2) Comparative test of different protein separation and purification methods

[0101] In the section 4.2 of the specific implementation case, we compared different protein purification methods. First, we used the organic solvent purification method, using acetone or isopropanol to reduce the solubility of the protein in the solution, thereby promoting protein precipitation and enzymatic hydrolysis; secondly, we used the FASP enzymatic hydrolysis method, using a 10kD ultrafiltration membrane to remove small molecule interfering substances, and then using trypsin for enzymatic hydrolysis; finally, the SP3 magnetic bead purification method, which uses the carboxyl groups on the magnetic beads to interact with protein molecules, and achieves protein binding and elution by changing the solution conditions (such as adding organic solvents), where the protein can be eluted directly on the magnetic beads.

[0102] Result analysis:

[0103] The samples used in the comparison case here are 10 μm thick mouse brain tissue slices, laser microdissection cut 10mm 2 The tissues (n=5) were lysed with RLTPlus lysis buffer and then the proteins were purified and analyzed by mass spectrometry using the above four different methods. The results are shown in Figure 5 and Figure 6 Note: The peptide loading amount here is 1 / 50 of each sample.

[0104] Figure 5 The following are the protein identification numbers using acetone precipitation, isopropanol precipitation, FASP method and SP3 magnetic bead method respectively. It can be seen from the figure that the protein identification number using SP3 magnetic bead method is the highest, while Figure 6 It can be seen that the overlap of the four methods is close to 70%, indicating that the types of proteins identified are similar and the technical repeatability is good.

[0105] Secondly, laser microdissection technology was used to collect tissues of different sizes on 10 μm thick sections of mouse brain tissue, and the method of the present invention was used to extract mRNA and protein from mouse brain tissues of different volumes. Since mRNA is easily degraded in small amounts in tissues and difficult to quantify, we performed reverse transcription and PCR (the number of cycles was uniformly set to 19) according to step 3.1 in the specific implementation case, and then quantified the obtained cDNA; the protein was enzymatically hydrolyzed into a peptide solution, and after the peptides were quantified, 200 ng was taken for mass spectrometry analysis. If it was less than 200 ng, it was drained and re-dissolved in 10 microliters of 0.1% formic acid solution before all were loaded onto the machine. The results are shown in Table 7. This method can be used in tissues as small as 0.01 mm 3 Sufficient cDNA was still obtained for subsequent research, and the number of protein identifications reached more than 3,000.

[0106] Table 7. Average number of protein identifications and average total amount of cDNA obtained at different cutting volumes

[0107]

[0108] This technology can be applied to fresh tissue, frozen tissue, etc. The technology is stable and has good application prospects.

Claims

1. A method for co-extraction of mRNA and protein from a trace sample, characterized in that: The following steps are involved: Oligod(T) magnetic beads were used to capture messenger RNA and separate it from protein, which was then purified using SP3 magnetic beads.

2. The method for co-extraction of mRNA and protein from a trace sample according to claim 1, characterized in that: Specific methods include: (1) Place tissue block slices on the laser microdissection stage, use laser microdissection technology to collect areas of interest on the tissue slices, and obtain traces of tissue of the same size; (2) placing the tissue from step (1) into a lysis solution for lysis; (3) After the lysis is completed, the mRNA is extracted by magnetic bead separation, reverse transcribed into cDNA and analyzed by real-time fluorescence quantitative PCR; (4) reducing and alkylating the protein in the lysate after extracting mRNA in step (3); purifying; (5) the protein purified in step (4) is dissolved in a mass spectrometry compatible lysis buffer and subjected to enzymatic hydrolysis; (6) Add formic acid solution to step (5) to acidify and terminate the reaction, freeze-dry, and re-dissolve to prepare a peptide solution.

3. The method for co-extraction of mRNA and protein from a trace sample according to claim 1 or 2, characterized in that: The lysis buffer in step (2) is selected from at least one of RLTplus lysis buffer, RIPA lysis buffer and Tris-HCl lysis buffer containing 1% SDS.

4. The method for co-extraction of mRNA and protein from a trace sample according to any one of claims 1 to 3, characterized in that: The cDNA in step (3) can also be used for library construction using the smart-seq2 method and transcriptome analysis using the second-generation sequencing technology.

5. The method for co-extraction of mRNA and protein from a trace sample according to any one of claims 1 to 4, characterized in that: The purification method in step (4) is selected from at least one of ultrafiltration membrane purification method, SP3 magnetic bead purification method and organic solvent precipitation method.

6. The method for co-extraction of mRNA and protein from a trace sample according to any one of claims 1 to 5, characterized in that: The mass spectrometry-compatible lysis solution in step (5) includes an ammonium bicarbonate solution.

7. The method for co-extraction of mRNA and protein from a trace sample according to any one of claims 1 to 5, characterized in that: The enzyme used in the enzymatic hydrolysis of step (5) includes Trpsin enzyme.

8. The method for co-extraction of mRNA and protein from a trace sample according to any one of claims 1 to 7, characterized in that: The trace peptide samples in step (6) were analyzed by liquid chromatography-mass spectrometry and subjected to whole proteomics quantitative analysis.

9. mRNA extracted using any one of the methods described in claims 1-8.

10. Protein extracted by the method according to any one of claims 1 to 8.

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