Differential display PCR
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Differential display PCR
Materials and Instruments
Reverse transcriptase Heat-stable DNA polymerase Anchored 3'-oligonucleotide primers Random 5'-oligonucleotide primers Total RNA Radiolabeled dATP Move I. Materials For more product details, please visit Aladdin Scientific website.
Amplification buffer DD-PCR Reverse transcription buffer DTT dNTP storage solution Placenta RNase inhibitor Sequencing gel loading buffer
Agarose Gel Electrolyte Gradient Sequencing Gel Shielded Tip Centrifuge Tubes Positive Displacement Pipettes PCR Instruments Water Baths
1. Buffers and solutions
10X amplification buffer
5X DD-PCR reverse transcription buffer (250 mmol/L Tris-Cl (pH 8.3), 375 mmol/L KCl, 15 mmol/L MgCl2 )
DTT ( 100 mmol/L)
4 dNTP storage solutions (20 mmol/L, pH 8.0)
Placental RNase inhibitor (20 units/ml)
5X Sequencing Gel Sampling Buffer
2. Enzyme and Buffer
Reverse transcriptase (RNA-dependent DNA polymerase)
The reverse transcriptase used in DD-PCR is an RNaseH activity-deficient enzyme (e.g., Superscript from Life Tech-nologies or StrataScript from Stratagene).
Heat Stable DNA Polymerase
3. Nucleic acids and oligonucleotides
The anchored 3'-oligonucleotide primer (300 μg/ml) was dissolved in 10 mmol/L Tris-Cl ( pH 7.6) containing 0.1 mmol/L EDTA
Random 5'-oligonucleotide primer (50 μg/ml) was dissolved in 10 mmol/L Tris-Cl (pH 7.6) containing 0.1 mmol/L EDTA.
Total RNA (100 μg/ml)
4. gel
Agarose gel
Electrolyte gradient sequencing gel
5. Radioactive material
Radiolabeled dATP (10 μCi/μl, specific activity 3000 Ci/mmol)
6. Specialized equipment
Shielded tips for automated micropipettes
Centrifuge tubes (0.5 ml, specialized for thin-walled amplification reactions)
Positive displacement pipettes
PCR instrument
Water temperature of the water bath is preset at 65°C and 94°C 
II. Methods
Optimization of DNA concentration: preparation of the first strand of cDNA
1. Using several 0.5 ml centrifuge tubes, set up a series of experimental reaction tubes to establish the optimal concentration of RNA in the control and experimental tubes, the so-called optimal concentration refers to the subsequent DD-PCR amplification of 100~300 bands on gel electrophoresis and radioautography. A series of 5-fold serial dilutions of the RNA samples with water was performed to produce a series of RNA sample concentrations ranging from 1 μg/ml to 100 μg/ml.
2. Select one or more primers from the Anchored 3'-Oligonucleotide Primer Library to set up a multiplexing reaction series with different dilutions of the RNA template:
RNA template 8.0 μl
Anchored 3'-oligonucleotide primer 2.0 μl
Reaction tubes were incubated at 65℃ for 10 min and then placed on a 37℃ water bath.
The amount of total RNA in the replication reaction should vary between 8 ng and 800 ng.
3. Add the following reagents for the multiplexing reaction:
5X DD-PCR Reverse Transcription Buffer 4 μl
100 mmol/L DDT 2 μl
200 μmol/L 4 dNTP mix 2 μl
Approx. 25 units/μl RNase inhibitor 0.25 μl
200 units/μl Reverse transcriptase 0.25 μl
H2O make up to 20 μl
Reaction tubes were incubated at 37°C for 1 h. The reaction tubes were incubated for 1 h at 37°C.
4. Heat the sample in the reaction tube at 94℃ for 10 min to inactivate the reverse transcriptase.
Optimizing RNA Concentration: Amplification and Preparation of Double-stranded cDNAs
5. Set up two experimental series of eight 0.5 ml amplification tubes. Each tube should contain the following reagents:
10X Amplification Buffer 2 μl
Anchored 3' oligonucleotide primer 2 μl
20 mmol/L 4 dNTP mix (pH 8.0) 1 μl
[ α-33P ] dATP or [ α-35S ] dATP ( 3000 Ci/mmol) 1 μl
5 units/μl Heat stabilized DNA polymerase 1 unit
H2O 9 μl
For each reaction tube, add 2 μl of 5' random primer and flick the tube wall to mix the reaction solution.
6. Take 3 μl of sample from each of the 8 tubes of the two series containing the reverse transcription of the test RNA and the control RNA, cap the tubes and gently mix the samples.
7. If the PCR instrument is not equipped with a heated lid, add a drop of mineral oil (~50 μl) to the top of the reaction mixture. Place the centrifuge tube on the PCR instrument.
8. The amplification reactions are denaturation, denaturation and polymerization (extension reactions); the corresponding cycling conditions and temperatures are listed below: 
9. At the end of the amplification program, remove the reaction tubes from the PCR instrument and add 5 μl of 5X Sequencing Gel Sampling Buffer to each tube.
10. Apply DNA Sequence Analysis type electrolyte gradient polyacrylamide gel electrophoresis to separate this radiolabeled amplicon Electrophoresis is carried out under a regulated power supply, and ends when the xylene cyanide blue electrophoresis reaches two-thirds of the length of the gel. The gel was drained and placed on the lower press of the radiolabeled autoradiographic negative.
11. Examine the DNA band profiles of the amplification reaction products from different concentration sources of control and test RNA. An ideal differential band profile should ideally clearly distinguish between 100 and 250 electrophoretic bands, and the optimal amount of DNA template will vary from sample to sample and from primer to primer. The optimal concentration of control and test RNA is chosen so that the maximum amount of DNA product is produced by primer pairing and guided amplification at that concentration.
Preparation of amplified cDNA for differential expression
12. Apply all primer combinations with the optimal amount of RNA template, recombination, reverse transcription and amplification reactions. All reaction cycles are designed to be set up on a PCR instrument using 96-well plates i.e. microtitre plates.
13. Apply polyacrylamide sequencing gel electrophoresis to separate the amplification reaction products as in steps 9 and 10. The amplification reaction samples obtained with each primer pair are upsampled on adjacent lanes of the sequencing gel, e.g., an amplification sample obtained from one RNA sample by applying the paired primers A + B is adjacent to another amplification product obtained from another RNA sample by applying the same pair of primers A + B on the gel lanes. Comparative studies of radioautographic band profiles are greatly simplified by numbering the samples in this way.
14. Comparative study of the band profiles of amplification products obtained from different RNA samples with each primer pair.
In characterizing different expression bands, it is often extremely important to repeat the experiment to determine the reproducibility of the initial finding. Although this precaution may not be actionable, ideally, different batches of samples of two RNAs should be applied simultaneously to facilitate corroboration.
Recovery and Reamplification of Differently Displayed cDNAs
15. Recover the target bands from the drained polyacrylamide gel. Prevent radiographic x-rays from being on top of the gel by marking the position of the target band on the radiographic x-ray with a soft pencil. Using a clean razor blade, cut the upper layer of radiographic film along with the lower layer of gel along the pencil markings to remove each destination band. Place a gel strip of the destination band on a piece of Whatman 3 MM paper; place each thin slice of drained gel paper into a 0.5 ml centrifuge tube (containing 50 μl of sterilized water).
16. Puncture the bottom of each overnight 0.5 ml centrifuge tube with a small needle; place each puncture tube into a 1.5 ml centrifuge tube and centrifuge for 20 s. Transfer the eluate to another larger centrifuge tube and discard the remaining gel strips in the amplicon tubes and the Whatman 3 MM paper strips.
17. Using the DNA fragments in the eluate as a template, add the following reagents one at a time to the amplification reaction tube:
10X Amplification Buffer 2 μl
2 μl of 10X Amplification Buffer 2 μl of DNA sample eluted from polyacrylamide gel
5'-random oligonucleotide primer 2 μl
3'-anchored oligonucleotide primer 2 μl
20 mmol/L 4 dNTP mix (pH 7.0) 1 μl
5 units/μl Taq heat-stable DNA polymerase 2 units
H2O 9.5 μl
18. If the PCR instrument is not equipped with a heated lid, add a drop of mineral oil (approx. 50 μl) to the top of the reaction mixture. Place the centrifuge tube above the PCR.
19. The amplification reactions are denaturation, denaturation and polymerization (extension reactions); the corresponding cycling conditions and temperatures are listed below: 
20. 5-10% of each re-amplified sample is taken and the amount of re-amplified DNA fragments is estimated by 1% (m/V) agarose gel electrophoresis.
21. If mineral oil is used to cover the top layer of sample liquid in the microcentrifuge tube (step 18), it can be removed by 150 μl of chloroform extraction at the end of the reaction.
22. Ligate the reamplified DNA fragments into a vector with a dT tail (e.g. Promega pEGM T vector) and transform the E. coli host bacteria with a portion of the ligand.
23. 6 or more transformed colonies are picked from a petri dish and their plasmid DNA is extracted and identified by digestion with the appropriate restriction endonuclease, and the recombinant transformants are identified by comparing the size of the inserted fragments.
24. Confirmation of the cDNA/mRNA molecules of differentially expressed candidates is performed by as many routes and methods as possible, including Northern hybridization, RNase protection, or quantitative PCR. mRNA in situ hybridization allows for tissue localization of tissues from a particular disease or different developmental stages and differentially expressed specific transcripts. 