In situ PCR

Summary

In situ PCR (in situ PCR) is a PCR reaction performed in tissue cells, which combines the advantages of in situ hybridization with cellular localization ability and highly specific and sensitive PCR technology to detect specific genomic sequences, transgenes and exogenous genes at the molecular and cellular levels, which is of great practical value for the study of the pathogenesis of diseases and clinical processes and pathological regression, and has great application prospects in the fields of molecular biology, cytology, molecular pathology and clinical diagnosis. It has great application prospects in the fields of molecular biology, cytology, molecular pathology and clinical diagnosis.

According to the different operation methods, in situ PCR can be divided into indirect and direct methods. The direct method has fewer steps, but has a higher false-positive rate; the indirect method has more steps and takes longer time, but the results are reliable.

Principle

According to the different operation methods, in situ PCR can be divided into indirect and direct methods.

The basic principle of the indirect method is to amplify specific nucleotide sequences in target cells by PCR after fixing tissues and cell specimens and digesting them with protease, and then combine them with in situ hybridization for nucleotide sequence detection and intracellular localization.

The basic principle of the direct method is to add labeled nucleotides or primers to the PCR reaction solution before performing in situ PCR. As amplification proceeds, the markers are directly incorporated into the PCR product, and then the target nucleic acid molecules are localized and detected intracellularly by radioautography, immunohistochemistry, or fluorescence detection.

Appliance

In situ PCR is commonly used in the study of disease pathogenesis, clinical process and regression of pathology, molecular biology, cytology, molecular pathology and clinical diagnostic fields.

Operation method

Indirect in situ PCR

Principle

The basic principle of indirect in situ PCR is to amplify specific nucleotide sequences in the target cells by PCR after fixing the tissue and cell specimens and digesting them with protease, and then combining them with in situ hybridization for the detection of nucleotide sequences and intracellular localization.

Materials and Instruments

Equipment:
①In situ PCR instrument
② constant temperature water bath
③Microscope
④Centrifuge
⑤ Siliconized slides and coverslips
⑥ micropipette (20 μl), tip (sterilized)
Reagents:
①Materials: cultured cells, specific nucleotide probes
② 10% formalin buffer, PBS, proteinase K and its buffer, TE
③ Upstream primer and downstream primer (20 μmol/L), dNTP mixture, Taq DNA polymerase, 10 x PCR reaction buffer, Klenow enzyme, Dig-dUTP, random primer
④ 0.2 mol／L EDTA (pH 8.0), 4 mol／L LiC1
⑤ Hybridization solution, hybridization buffer I, hybridization buffer II, hybridization buffer II
⑥Color development solution
⑦Sheep anti-digoxin antibody-alkaline phosphatase conjugate (1:500), sheep serum
⑧ Triton X-100, 20 x SSC, 4 x SSC, 2 x SSC and 0.1 x SSC
⑨ Anhydrous ethanol, 70% ethanol
⑩ sterile paraffin oil, silicone oil, xylene

Move

The basic process of indirect in situ PCR can be divided into the following steps:

(i) Reagent preparation

(1) 10% formalin buffer.

(2) Proteinase K Dissolve proteinase K powder with sterilized 50 mmol/L Tris (pH 8.0), 1.5 mmol/L calcium acetate, and prepare a solution with a concentration of 20 mg/ml, store at -20 ℃, and work at a concentration of 0.25 mg/ml.

(3) dNTP mixture of dATP, dGTP, dCTP and dTTP sodium salt of 100 mg each combined, add 2 ml of deionized water to dissolve, with 0.1 mol / L NaOH pH adjustment to 7.0 ~ 7.5, so that the concentration of 5 mmol / L, divided into packages -20 ℃ storage.

The commercialized mixture (2 mmol/L each) is also available.

(4) Taq DNA polymerase 5 U/μl, the final concentration of which is 3 U in a 50 μl reaction volume.

(5) PCR reaction buffer (10x) 500 mmol/L KC1, 100 mmol/L Tris-HCl (pH 8.4), 15 mmol/L MgCl₂, 0.5% Tween-20, 1 mg/L BSA.

(6) Upstream and downstream primers According to the nucleotide sequences to be detected, the most suitable upstream and downstream primers were designed by the primer design software, and synthesized by the DNA synthesizer at a concentration of 20 μmol/L. The primers were used for the detection of the nucleotide sequences, and the primer design software was used to design the most suitable upstream and downstream primers.

(7) Klenow enzyme 5 U/μl.

(8) 0.2 mol/L EDTA pH 8.0.

(9) 4 mol/L LiCI.

(10) TE 10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0.

(11) Hybridization solution 50% deionized formamide, 4 X SSC, 10% dextran sulfate, 0.5 ng/μl digoxigenin-labeled probe.

(12) Hybridization Buffer I 100 mmol/L Tris-HC1, 150 mmol/L NaCl, pH 7.5.

(13) Hybridization buffer II 100 mmol/L Tris-HCI, 100 mmol/L NaCl, 50 mmol/L MgCl₂, pH 9.5.

(14) Hybridization buffer III 10 mmol/L Tris-HCI, 1 mmol/L EDTA, pH 8.0.

(15) Color development solution Nitrobenzotetrazolium salt (NBT) 45 μl, 5-bromo-4-chloro-3-indole phosphate 35 μl, levamisole 2.4 mg, add buffer II to 10 ml.

(16) 4 x SSC, 2 x SSC and 0.1 x SSC were diluted with 20 x SSC (20 x SSC: 3 mol/L NaCl, 0.3 ml/L sodium citrate, pH adjusted to 7.0 with 1 mol/L HCl).

(ii) Treatment of cultured cells

(1) Wash the cells in the Petri dish with PBS directly once, add 10% formalin buffer and leave overnight, centrifuge at 2000 r/min for 5 min, resuspend the cells with 5 ml PBS, and pipette 50 μl onto the slide.

(2) Take 3 slides containing cell suspension and silicone them with silicone oil, this process is necessary for the adsorption of cells.

(3) Protease digestion: add proteinase K and its buffer, the working concentration of proteinase K is 0.25 mg/ml, the digestion time is 10-15 min at room temperature.

(4) Wash away the digestive enzymes with distilled water, and then heat the slide to 95-100 ℃ for 2 min to inactivate the digestive enzymes.

(C) PCR reaction

(1) After the slide is cooled, add 25 μl of PCR reaction solution: 2.5 μl of 10 x PCR buffer, 2.5 μl of dNTP (200 μmol/L for each nucleotide), and 1 μl of each of the upstream primer and downstream primer.

When the coverslip was heated to 65-80 ℃ on the PCR instrument, one corner of the coverslip was immediately removed, and 1.5 U of Taq DNA polymerase was added into the coverslip, which was called "hot start" method.

(2) Immediately add drops of preheated paraffin oil around and on the coverslip to fix the coverslip.

(3) The PCR reaction was performed using an in situ PCR instrument: denaturation at 94 ℃ for 3 min, 94 ℃ for 1 min, 55 ℃ for 2 min, 72 ℃ for 1 min, 30 cycles.

(4) After PCR was completed, paraffin oil was washed with xylene for 5 min to remove paraffin oil, and then washed with anhydrous ethanol for 5 min and air dried.

(D) Probe labeling

Add about 1 μg of denatured specific nucleotide probe, 2 μl of random primer, 2 μl of dNTP and Dig-UTP to the reaction solution, add 5 U Klenow enzyme after mixing, and make up 50 μl of sterile double-distilled water.

The reaction was terminated by adding 2 μl of 0.2 mol/L EDTA (pH 8.0), 2.5 μl of 4 mol/L LiCl and 75 μl of -20 ℃ pre-cooled anhydrous ethanol, and then placed at -20 ℃ for 2 h. After centrifugation at 12,000 g for 20 min, the precipitate was washed with pre-cooled 70% ethanol, centrifuged and dried, and then dissolved in TE and stored at -20 ℃.

(E) In situ hybridization

(1) Add 10-30 μl of hybridization solution (prehybridization solution plus probe) on the slide, cover the slide and seal the edge with paraffin oil.

(2) Denature at 94 ℃ for 8 min and hybridize at 42 ℃ overnight.

(3) After completion of hybridization, paraffin oil was washed off with xylene, and then anhydrous ethanol was used to remove benzene.

(4) Remove the coverslip from the slide in 2 x SSC, and then wash with 4 x SSC, 42 ℃ for 3 times, 5 min each time; 0.1 x SSC, 42 ℃ for 3 times, 5 min each time.

(VI) Color development

The slides were washed with buffer I for 1 min, and buffer I containing 2% sheep serum and 3% Triton X-100 was incubated at 37 ℃ for 30 min, dropwise addition of sheep anti-digoxigenin antibody-alkaline phosphatase conjugate at room temperature for 3-5 h. The slides were washed with buffer I for 1 min, and buffer I containing 2% sheep serum and 3% Triton X-100 was incubated at 37 ℃ for 30 min.

Buffer I was washed for 10 min, buffer II was washed for 10 min, and the slides were sealed in a dark box containing color development solution at light-avoidance, incubated for 2~4 h, and the reaction was terminated by immersion in buffer II.

(G) Experimental results and analysis Positive reaction was defined as purple-blue precipitation under microscope.

Caveat

(1) Cell fixation Not all fixatives can be successfully used for in situ PCR. the in situ PCR products of cells fixed in ethanol or acetone are often free in the reactants, reflecting the failure of these fixatives to denature and cross-link proteins and nucleic acids.On the contrary, 10% neutral formalin could well coagulate proteins and cross-link nucleic acids, thus forming an effective network barrier to limit the diffusion of PCR products. The fixation time should not exceed 15 hours.(2) Protease digestion Proper protease digestion is beneficial to establish the channel for PCR reagents to contact the target DNA and to fully expose the target DNA.However, since protease digestion will destroy the protein-nucleic acid network already formed, and excessive digestion will make the PCR product easy to diffuse, the digestion time should be controlled.(3) In the process of in situ PCR, the full exposure of target sequences and the maximization of reagents into the cell should be the key to grasp.In the in situ PCR reaction system, primers, Taq DNA polymerase and Mg2+concentration should be higher than that of conventional liquid-phase PCR, especially Mg2＋concentration.(4) Primer design and selection In addition to the general principles of primer design for PCR reaction, the primers selected for in situ PCR, the length of the amplification product should not be too short, otherwise it is easy to diffuse; nor should it be too long, otherwise it will affect the amplification efficiency.(5) Reduce primer mismatch Adopt the hot start method, in the PCR reaction, often at a relatively high temperature and then add Taq enzyme or primer, that is, "hot start" method.This method can greatly reduce the synthesis of non-specific DNA, reduce the possibility of primer mismatch, high sensitivity and specificity.(6) Cycle cycle The amplification efficiency of in situ PCR is not as good as that of liquid-phase PCR, therefore, the cycle cycle should not be too small, otherwise, there are fewer objects and the signal is too small; however, if the cycle is too large, the product will diffuse and a large number of non-specific DNA synthesis.Therefore, the cycle usually used is 20 to 30.(7) The number of digoxigenin-labeled probes labeled by in situ hybridization should not be less than 50%, and the concentration of the probe in the hybridization solution should not be less than 5 pmol/ml, and if it is too low, the hybridization signal will be weak. The hybridization temperature should be calculated according to the nucleotide (G + C) content in the probe.(8) Negative control The in situ PCR reaction is prone to false positives or false negatives, for this reason it is necessary to set up appropriate controls. Digesting the control with DNA or RNA enzymes, omitting primers, polymerase and probes and other negative controls is necessary.Using negative or positive specimens for each process ensures that all reagents and processes are appropriate.

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