single-cell PCR

Summary

Single-cell PCR utilizes flow cytometry to isolate a specific type of cell. Using a single cell, PCR can be analyzed at the DNA or mRNA level to find molecular alterations within a single cell. Currently, there is one main method used for single-cell PCR: single-cell PCR.

Principle

The basic principle of single-cell PCR is that individual cells often contain very little (as little as one copy) of the target DNA or RNA, and therefore specific conditions are required to amplify DNA or RNA sequences from individual cells. The first step is to isolate a specific cell, which can be accomplished using microscopic manipulation (for cells with distinctive cellular morphology) or flow cytometry.

After isolation of the individual cells, the cells are lysed to release the target DNA or RNA; if DNA is analyzed, it is preferable to lysed the cells without destroying the nucleus, and then use the lysate as a template for the PCR reaction. To analyze RNA, reverse transcribe the RNA into cDNA and then perform PCR.

The PCR product can be analyzed by agarose gel electrophoresis directly or by further dot hybridization or Southern blot.

Appliance

Single-cell PCR is commonly used in the following applications: 1. Single-cell PCR is used for typing single spermatozoa. Using single-cell PCR to analyze the frequency of allelic exchanges of interlinked genetic markers in a single spermatozoa, the frequency of recombination between neighboring genetic markers can be calculated, and the distance between interlinked genetic markers can thus be estimated, which provides a powerful method for constructing the genetic map of mammal genome. 2. Single-cell PCR is used in the study of lymph node hematopoietic system diseases. Single sperm typing is particularly effective for genetic mapping of species that cannot reproduce in large numbers or have exceptionally long generation cycles. 2. Single-cell PCR has been applied to the study of lymph node hematopoietic disorders, such as the study of the origin of Reed-Sternberg cells (RS cells) in Hodgkin's disease. Since the tumorigenic component of Reed-Sternberg cells in lymph nodes invaded by Hodgkin's disease is often less than 1%, the DNA extracted from the tissues is actually a mixture of tumor cell DNA and DNA from other cells, and this extraction method is therefore not suitable for the detection of RS cells. The combination of immunohistochemistry and in situ hybridization allows the detection of gene expression in individual Hodgkin cells and RS cells, but does not allow a more detailed study of their DNA. Single-cell PCR remedies these shortcomings: Kuppers et al. isolated RS cells from a patient with sclerosing Hodgkin's disease and used single-cell PCR to obtain 12 IgH gene rearrangement products, of which 8 were sequenced and 7 had identical sequences, suggesting that at least some of the RS cells in this patient had clonal B-cell origin. suggests that at least some of the RS cells in this patient with sclerosing Hodgkin's disease were derived from clonal B cells. The methodological feasibility of single cell isolation and PCR on tissue sections is well established, and it is very suitable for the study of lymphopoietic lesions. Single-cell studies can also be applied to the study of lymph node growth centers and other types of malignant lymphomas, since information on clonal correlations, intraclonal differences, and perpetuation of mutations can only be obtained by single-cell studies. Modifications of this method can be used for a variety of other tumors, especially for oncogene and oncogene analysis of samples with large numbers of non-tumor cells intermingled with the tumor cells, and for clonality studies of the tumor cells.

Operation method

Single-cell PCR

Principle

The basic principle of single-cell PCR is to isolate a single cell and then lysed the cell to release the target DNA or RNA. If analyzing DNA, it is preferable to lysed the cell without destroying the nucleus, and then use the lysed product as a template for the PCR reaction. To analyze RNA, reverse transcribe the RNA into cDNA and then perform the PCR reaction.

Materials and Instruments

Equipments: Flow cytometer, PCR instrument, centrifuge, electrophoresis machine
Reagents:
1, reagents for single cell separation:
Taipan blue, PBS, monoclonal antibody, colloidal gold, 10% Dimethyl Sulfoxide (DMSO), 0.9% NaCl solution, 0.5% trypsin, 0.04 mg/ml DNA enzyme, 0.16% Hydroxypropyl Methyl Cellulose (HPMC), Alkaline Phosphatase-Fast Red, Hematoxylin.
2. Single-cell PCR reagents:
A. 1 × PCR reaction buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl pH 8.3, 2.5
mmol/L MgCl
2
, 0.1 mg/ml gelatin)
B. 0.05 mg/ml Proteinase K
C. 20 mmol/L DTT
D. 1.7 μmol/L SDS
E. PCR primers
F. dNTP
G. Taq DNA polymerase
3. Reagents for analyzing single-cell PCR products:
A. SSC buffer
B. 50% deionized formamide
C. 5 × Denhardt's solution
D. Salmon sperm DNA
E. SDS
F. 0.1% Na
2
H
2
P
2
O
7
G. SET buffer

Move

The basic process of single-cell PCR can be divided into the following steps:

(I) Separation of single cells

Isolation of single lymphocytes: Dilute 10 ml of blood with the same volume of RPMI1640 medium in a 50 ml centrifuge tube, cover the top layer with 20 ml of Ficoll-Paque Plus that has been placed at room temperature, centrifuge at 500 g for 20 min at room temperature, carefully aspirate the middle dark yellow layer of liquid, and place it in a 50 ml centrifuge tube. Resuspend the cells in 40 ml of PBS, centrifuge at 500 g for 15 min, discard the supernatant and resuspend the cells in 10 ml of PBS. Take 10 μl of cell suspension, add 15 μl of PBS and 25 μl of Taipan blue for cell counting. After centrifugation at 300 g for 5 min, the cell suspension was resuspended in PBS to a concentration of 2 × ¹⁰⁷ cells/ml. 50 μl of cell suspension was placed in a 5 ml centrifuge tube, 20 μl of undiluted monoclonal antibody (e.g., anti-CD19) was added, and the tube was placed at 4 ℃ and protected from light for 30 min. The cells were washed twice with 4 ml of PBS, and centrifuged twice at 120 g for 5 min each time. The cells were washed twice with 4 ml PBS and centrifuged at 120 g for 5 min each time. The cells were sorted by flow cytometry and stored at -80 ℃.

Isolation of individual cells from fresh tissues is illustrated by the example of rat retinal ganglion cells:

(1) Retrograde labeling of retinal ganglion cells: Before surgical dissection, the test animals were first intraperitoneally injected with a mixture of Rompun and Ketalar at doses of 10~15 mg/kg and 30~100 mg/kg body weight, respectively. Retinal ganglion cells were retrogradely labeled from the superior colliculus with a fluorescent tracer, colloidal gold. When exposed, a gelatin sponge absorbing 3% colloidal gold and 10% dimethyl sulfoxide (DMSO) in a 0.9% NaCl solution was placed over the surface of the superior colliculus. In this way cell ends will be exposed to colloidal gold and retrogradely transferred to neuronal cell bodies in the retina. Labeling was optimized by adding colloidal gold for 7 d. Animals were killed by intraperitoneal injection of 3 to 4 ml of phenobarbital. The temporal-nasal margins around the eyes were marked with sutures, and the eyes were removed and quickly placed on ice in preparation for dissection.

(2) Mechanical layering to isolate retinal ganglion cells: The eyeballs were separated in sterile PBS buffer, and the retinas were carefully detached from the sclera and divided into four parts. A piece of retina was placed on a nitrocellulose membrane (5 mm × 5 mm) with forceps, with the photoreceptor facing the nitrocellulose membrane. The vitreous humor was removed, and the nitrocellulose membrane and retina were placed in PBS containing 0.5% trypsin and 0.04 mg/ml DNAase at 37 ℃ for 15 min. The nitrocellulose membrane was placed on Millipore's filter paper for 5 s, the excess liquid was aspirated, and the inner layer of the retina was placed face down on an uncoated coverslip (24 mm × 60 mm). A slightly smaller coverslip (24 mm × 32 mm) was placed on the Millipore filter paper to promote adhesion to the glass surface, forming a "sandwich" structure, and placed at 37 ℃ for 5 min. The small coverslip was removed, and the separated layers were obtained by carefully lifting up the filter paper and the retina with forceps. The separated cells were immediately placed in PBS containing 0.16% hydroxypropyl methylcellulose (HPMC) to increase the viscosity and stabilize the cells.

(3) Collection of individual retinal ganglion cells: Cells are kept on an initial coverslip and picked under an inverted fluorescence microscope. Individual retinal ganglion cells can be distinguished by the light emitted by colloidal gold and are aspirated in PCR tubes using a hand-controlled micro-sampler.

3, Isolate individual cells from tissue sections and freeze sections 5~10 μm thick, dry overnight, fix in acetone for 10 min on the next day, dry for 20 min, dropwise add appropriate monoclonal antibody, immunohistochemistry staining according to the ABC method, using alkaline phosphatase-fast red chromatography, hematoxylin re-staining. On the stained sections, 0.01 mol/L TBS (pH 7.4) buffer was added dropwise. The desired cells were first located under light microscope with 20× objective × 10× eyepiece, and then the desired cells were carefully separated from the surrounding cells under 60× objective × 10× eyepiece using a separating micropipette with a diameter of 1 to 2 μm drawn from a hard glass microelectrode blank (Narishige, Japan). The cells were then fed into a 10-20 μm diameter aspiration micropipette made from a hard glass microelectrode blank, and the cells were drawn into the PCR tubes by a hydraulic actuator. The cells were sucked into the PCR tubes by a hydraulic actuator and stored at -20 ℃.

(ii) Single-cell PCR

① Transfer single cells into a PCR tube with 20 μl of lysis buffer. It contains 1 × PCR reaction buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl pH 8.3, 2.5 mmol/L MgCl₂, 0.1 mg/ml gelatin) and 0.05 mg/ml Protease K, 20 mmol/L DTT, 1.7 μmol/L SDS.

② After a warm bath at 37 ℃ for 1 h, the sample was heated to 85 ℃.

③ Add the samples to 100 μl of PCR reaction system, including 1 × PCR reaction buffer, 1 μmol/L PCR primers (1 μmol/L each), 187.5 μmol/L dNTP (dATP, dCTP, dGTP, dTTP, 187.5 μmol/L each), 100 ng of template DNA and 2 U of Taq DNA polymerase. DNA polymerase.

The PCR reaction conditions were designed according to the annealing temperature of the primers and the expected fragment size. The standard reaction conditions were as follows: 95 ℃, denaturation for 5 min; 95 ℃, 30 s, 55 ℃ (depending on the annealing temperature of the PCR primers), 30 s, 72 ℃, 40 s (depending on the expected fragment size of the PCR), 30-50 cycles; and 72 ℃ for 10 min.

(iii) Analysis of single-cell PCR products

The PCR products were directly analyzed by agarose gel electrophoresis. After electrophoresis, the products were stained with EB and analyzed under ultraviolet light. Under most conditions, the products from one round of PCR reaction are not sufficiently visualized by EB staining and need to be further analyzed by either dot hybridization or Southern blot.

1、Dot hybridization

(1) Preparation of sample membrane

Pre-denaturation of DNA samples: Dissolve DNA in water or TE, boil for 5~10 min, and then rapidly cool in an ice bath to denature the sample.

② Nylon membrane pretreatment wear clean gloves, cut the nylon membrane to the appropriate size as needed, and cut off a corner as the order of sampling mark. If manual spotting is used, use a pencil to mark small cells on the nylon membrane according to the area of 0.8~1.0 ^cm2. Wet it with distilled water and then immerse it in 6×SSC for at least 30 min, then remove the membrane and wind it for use.

Spotting can be done directly by hand or with a vacuum filtration sampler (spot or slit), depending on the situation. Manual direct dispensing: Use a micropipette to dispense the denatured nucleic acid sample onto the labeled spots on the nylon membrane. The diameter of the spot should not be too large and should be within 0.5 ^cm2. Spot a single sample several times in small amounts, and air-dry the sample while spotting.

Spot Vacuum Filtration Sampler Spotting:

a. After cleaning the sampler in the conventional way, wash the sampler with 0.1 mol/L NaOH and rinse it well with sterile triple-distilled water;

b. Wet the nylon membrane and cover it on the support pad of the sampler (or pre-moistened filter paper), carefully remove the air bubbles, use Parafilm to close the parts not covered by the nylon membrane, re-install the sampler, and turn on the vacuum pump;

c. Fill the spiking hole with 10 x SSC, pump until all liquid is drained, turn off the vacuum pump, and repeat once;

d. Add 2 times the volume of 20×SSC to the above pre-denatured sample and add it to each well, vacuum filtration, after all the liquid is drained, add 10×SSC again and filtration twice. e. Wait for 10×SSC to be drained, then add 10×SSC to each well;

e. After 10×SSC filtration, continue to maintain the vacuum for 5 min, so that the nylon membrane is dry.

④Fixation: Place the sample membrane on the filter paper after the sample counting, air-dry it naturally at room temperature, and then bake it under vacuum at 80 ℃ for 2 h to fix the nucleic acid samples. Fixed sample membrane, sealed in a plastic bag for use (-20 ℃ can be stored for several months).

(2) Pre-hybridization: Wet the sample membrane with 2×SSC, put it into a hybridization tube, add 10-20 ml of pre-hybridization solution, and incubate it at 42 ℃ for 2-4 h in the hybridization oven.

The final concentration of each component of the prehybridization solution was 6×SSC, 50% deionized formamide, 5×Denhardt's solution, 0.5 mg/ml salmon DNA, 0.5% SDS.

(3) Hybridization

① Prepare hybridization solution (final concentration): 6×SSC, 5×Denhardt liquid, 50% deionized formamide, 0.1 mg/ml salmon DNA, 0.5% SDS.

② Probe denaturation: The radioactively labeled double-stranded DNA probe needs to be denatured. Generally, the DNA probe is boiled in a boiling water bath for 5 min, and then quickly placed in an ice bath.

③Hybridization: Remove the hybridization tube from the hybridization oven, discard the pre-hybridization solution and add 5~10 ml of hybridization solution. Add the radioactively labeled probe, carefully remove air bubbles, and incubate at 42 ℃ for 16-20 h in general.

(4) Wash the membrane after hybridization, pour the hybridization solution into the radioactive waste container, take out the sample membrane, put it into a plate containing 2×SSC/0.1% SDS, and rinse it with shaking at room temperature for 5 min. 2×SSC/0.1% SDS should be washed twice at room temperature, each time for 15 min, 0.1×SSC/0.1% SDS should be washed twice at room temperature, each time for 15 min, 0.1×SSC/0.1% SDS should be washed twice at room temperature, each time for 15 min, 0.1×SSC/0.1% SDS at 55 ℃, and 0.1×SSC/0.1% SDS at 55 ℃. 0.1% SDS 55 ℃ washed twice, each time 15 min.

(5) Radiation autoradiography sample membrane after rinsing, placed on a clean filter paper, absorb excess water on the membrane, wrapped in a layer of plastic wrap. Place the sample film in a dark box, press an X-ray film on top of the sample film, and after radiographic autoradiography at -80 ℃ for 24~48 h, rinse the X-ray film according to the routine: develop for 1~5 min; fix for 5 min; rinse under running water for 10 min, and dry naturally.

(6) Observation of results: The presence or absence of the target gene and the amount of the target gene can be determined by the presence or absence and intensity of the exposure point. Using the automatic gray scale scanner to scan the exposure points and calculate the integrated optical density value, semi-quantitative analysis can be carried out.

Southern blot

① Slow electrophoresis (1 V/cm) of PCR products on agarose gel (0.65%-0.8%) for 24-48 h. After electrophoresis, the products are analyzed by UV analysis.

(ii) After electrophoresis, the gel is photographed under ultraviolet light and a transparent fluorescent ruler is placed along the edge of the gel so that the migration distance of the DNA reference can be read from the photograph.

(iii) Soak the gel in several times the volume of 0.25 mol/L HCI for 20 min and shake gently and continuously.

④ Soak the gel in several times the volume of denaturing buffer (1.5 mol/L NaCl, 0.5 N NaOH) for 30 min with gentle shaking.

⑤ Discard the denaturing buffer, add several times the volume of neutralizing buffer (1 mol/L Tris HCI pH7.4, 1.5 mol/L NaCI), and shake continuously for 30 min at room temperature.

(vi) The gel was placed in 20×SSPE for 30 min at room temperature.

(vii) Electrotransfer the DNA from the agarose gel to the nitrocellulose filter membrane by capillary transfer or electrotransfer method.

⑧ After the end of the transfer, use a pencil to mark the position of the gel spiking hole.

⑨ UV irradiation crosslinking (120 ^mJ/cm2 ).

⑩ 2×SSPE rinsed at room temperature.

⑪ Place the filter membrane in the center of two sets of 3 MM filter paper, and dry bake it in a vacuum oven at 80 ℃ for 1 h. ⑪ Place the filter membrane in the center of two sets of 3 MM filter paper.

⑫ Place the membranes in prehybridization solution (10×Denhard's, 4X SET, 0.1% SDS, 0.1% Na2H2P2O7, 100 pg/ml denatured salmon sperm DNA) and incubate at 65 ℃ for 1 h in the hybridization flask.

The DNA probe and salmon DNA were heated at 100 ℃ for 5 min to denature the DNA probe and salmon DNA, and then quickly placed on ice for 5 min.

⑮ Take 200 μl of DNA probe and 100 μl (10 mg/ml) of salmon DNA and add it to 10 ml of new prehybridization solution. Discard the prehybridization solution from the hybridization vial and add the hybridization solution containing the denatured probe. hybridize at 65 °C overnight.

⑯ Transfer the membrane to a rinse buffer (0.4 × SET, 0.1% SDS, 0.1% Na2H2P2O7) containing several hundred ml of rinsing buffer, and rinse the membrane twice for 10 min each in a 65 °C water bath shaker with gentle shaking.

⑰ Place the filter membrane in two sheets of 3 MM paper to dry slightly, wrap the membrane in Saran wrap, and attach several fluorescent labels for later calibration of the radiographic autoradiography to the position of the membrane.

⑱ Place the film in an X-ray fixture and expose at -70 °C for 12-48 h with an intensifying screen.

⑲ Rinse the X-ray film as usual: develop for 1-5 min; fix for 5 min; rinse under running water for 10 min; dry naturally.

⑳ The presence or absence of the target gene and the amount of the target gene can be determined by the presence or absence and strength of the exposure point. The exposure points are scanned with an automatic grayscale scanner, and the integrated optical density value is calculated for semi-quantitative analysis. Attachment: 20 X SET buffer: 3 mol/L, NaCl 0.4 mol/L, Tris-HCl, pH 7.5, 20 mmol/L EDTA

3. Single-cell RT-PCR

① Melt the PCR tubes containing single cells and add 3 μl of 5% NP-40 and 1 μl of 15 mmol/L reverse transcription primer or oligodT primer of the gene to be analyzed on ice. The PCR tubes were heated to 65 ℃ for 3 min, cooled at 25 ℃ for 3 min, and then put on ice to cool.

② Add 2 μl of 10× reverse transcription buffer, 2 μl of DTT, 1 μl of 10 mol/L dNTP and 0.5 μl of reverse transcriptase (e.g. Superscript II RT) to the PCR tube, and add RNase-free water to a final volume of 19.5 μl.

③ Place the PCR tube at 37 ℃ and react for 1 h. After synthesizing the first cDNA strand, heat to 70 ℃ and keep it at 10 min to inactivate reverse transcriptase.

④ Use the cDNA mixture obtained in step 3 as template for PCR reaction. In the PCR tube, mix the following solution: cDNA mixture 8 μm.

cDNA mixture 8 μl

20 μmol/L upstream primer 0.5 μl

20 μmol/L downstream primer 0.5 μl

10×Pfu PCR Reaction Buffer 6 μl

5 U/μl Pfu polymerase 1 μl

10 mmol/L dNTP 1.6 μl

Add water to a final volume of 60 μl.

⑤ Perform PCR reaction according to the standard PCR reaction program. In a typical single-cell RT-PCR reaction, two rounds of PCR are performed. For the second round of PCR, the product of the first round of PCR is used as the template for the PCR reaction, and the primer inside the primer of the first round of PCR is designed for the nested PCR reaction.

(vi) Analysis of single-cell RT-PCR products. The products of the first or second round of PCR reactions are electrophoresed on a 1% agarose gel, purified and recovered using the QIAquick Gel Recovery Kit, and sequenced. The sequences were compared and analyzed with those in the database. For semi-quantitative analysis, the PCR products can be analyzed by dot hybridization or Southern Blot at the end of the first PCR reaction.

Caveat

① Although single-cell PCR has many advantages, it is difficult to operate, requires a high level of laboratory work, and has a very large workload, so it can only be carried out by a small number of organizations at present. First of all, since the research object is a single cell, it is necessary to strictly prevent contamination. Therefore, in addition to strict sterilization of experimental equipment, as well as set up a variety of controls to eliminate interference, the operator should also pay attention to the operator can not bring items that can cause contamination into the single-cell operation room.In general, single-cell PCR should be repeated and double-blind. The following specific measures should be taken:a. Before entering the single-cell room, the operator must take a shower, then change into the special uniform and working shoes in the preparation room, and wear gloves, and pay particular attention to the fact that items from other laboratories are not allowed to be brought into the single-cell room;b. All experimental items such as gun tips, pipettes, reagents, etc. must be for the exclusive use of the single-cell room;c. Micropipettes for single-cell extraction must be sterilized at high temperature and replaced every time a cell is selected;d. Immunohistochemistry must also be performed in a dedicated laboratory, with the same requirements as entering the single-cell room;d. Immunohistochemistry must also be performed in a dedicated laboratory with the same requirements as for single-cell rooms. e. Preparation of reagents, addition of templates, PCR reactions, and electrophoretic detection of PCR products prior to the PCR reaction must be carried out in separate rooms;f. Frequent changing of gloves (especially after contact with DNA templates and PCR products), use of aerosol-proof disposable pipette tips etc;g. Setting up a buffer control, i.e., aspirating only the buffer covering the sections and not the cellular component, in which case PCR amplification should also be completely negative, and the presence of a positive band is evidence of cellular contamination in the buffer covering the sections.② Single-cell extraction is also difficult because it requires a very high degree of accuracy, which depends on the precision of the instrumentation and the level of the operator. Contamination of neighboring cells should be avoided to the greatest extent possible. After single-cell extraction, PCR reactions and DNA sequencing are often required. This places high demands on the amount and purity of the template. Compared with the conventional use of whole tissue DNA extraction, the purity of single-cell DNA is significantly higher, but the amount is correspondingly lower, resulting in the amplification efficiency of single-cell PCR is lower than that of whole-tissue PCR, which increases the workload.The amplification efficiency of single-cell PCR is lower than that of whole-tissue PCR because of the following reasons:a. When separating single cells from tissue sections, the nuclei of single cells are partially lost in 5-10 μm frozen sections, and the larger the cells are, the more components are lost, so the compensatory measures are to increase the thickness of frozen sections to ensure the integrity of the nuclei of the cells as much as possible.b. Single-cell operation is not allowed to use phenol, chloroform extraction, because it will cause the loss of the already very small amount of DNA, the use of proteinase K digestion, and then inactivate proteinase K method, but this purification method can not guarantee that the DNA of each cell to meet the requirements of PCR amplificationc. When aspirating cells, the tip of the micropipette will cause inward flow of buffer due to capillary adsorption. If the inward flow is not well controlled, it will increase and lead to a change in the buffer concentration in the whole system, which will also lead to a lack of positive results.d. The process of slicing, immunohistochemistry, and single-cell extraction takes a long time and is complicated, which may cause damage to cell DNA. All of the above results in single-cell amplification efficiencies of up to about 50% of the whole tissue.

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