Differential Display Technology (DD) Experiment

Summary

D D was first proposed by Liang and Pardee in 1992. To date, about 1700 articles related to DD have been published, which shows its great influence. Many genes associated with neurodegeneration and apoptosis have also been identified by the DD approach. The principle of DD is based on the analysis of cDs.

The principle of DD is based on the random amplification of cDNA molecules and their subsequent isolation by size. For this purpose, total RNA from target cells or tissues needs to be first isolated and reverse transcribed into cDNA.

Modern Neuroscience Research Techniques

Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun

Operation method

Differential display experiment

Materials and Instruments

Sterile glassware Sterile Eppendorf tubes Eppendorf tips Sterile falcon centrifuge tubes
4 mol L guanidine isothiocyanate
RNA extraction kit Eppendorf centrifuge Gel electrophoresis unit Direct blotting unit DNA autosequencer Water bath Spectrophotometer Bowl

Move

I. Isolation of RNA 1. General principles

The most critical factor in the success of differential display technology is the quality of the RNA. In order to minimize the degradation of RNA should be done:

-Wear gloves

Use sterile utensils. All plasticware should be left at 180°C overnight.

Use RNAase-free glassware. All glassware must be overnighted at 180 L. All buffers must have RNAase removed. Therefore, DEPC-treated redistilled water should be used (2.5 ml of DEPC force-ported into 2.5 L of redistilled water under high pressure)

-Operation on ice

2. Tissue homogenization

1. Tissue. Prior to manipulation at -70X: Freeze tissue and weigh. Grind frozen tissue immersed in liquid nitrogen to a powder in a mortar and pestle. Wear insulated gloves and protective eyewear to avoid damage from liquid nitrogen. Be sure to completely immerse the tissue in liquid nitrogen, as the damaged cells will release large amounts of RNAase at this point. Use a funnel to transfer the ground tissue from the mortar to a 50 ml sterile plastic Falcon tube. As soon as the liquid nitrogen evaporates, i.e., I ml of TRIzol is added to each 50-100 mg of tissue, shaking vigorously until well mixed. When all the tissue is completely dissolved in TRIzol, the RNAase activity is completely inhibited by GTC. At this stage, the solution can be stored at 4C overnight. However, we recommend proceeding immediately to the next step of the treatment until a complete absence of RNAase is achieved.

2. Monolayer cells. Cells are lysed by force-feeding Iml of TRIzol directly into a petri dish (3.5 cm diameter). The resulting solution is then transferred to a 2.0 ml Eppendorf tube.

3. Suspend the cells. centrifuge at IOOOg? for 5 min. add ImlTRIzol per 0.5Xl06~l.OX106 of eukaryotic cells with vigorous shaking.

3.Extraction

1. Incubate the RNA-containing TRIzol solution at room temperature for 5 min.

2. Force 0?2 ml of chloroform into each ml of TRlzol.

3. Shake for 15s, let stand at room temperature for 30s, and shake again for 15s.

4. Transfer the solution into a 2?0 ml Eppendorf tube.

Centrifuge at 15OOOg for 15 min at 5.4°C.

4.RNA precipitation

1. After centrifugation, transfer the upper colorless aqueous phase into a new Eppendorf tube.

Note: Avoid mixing intermediate phase substances. These substances contain proteins and genomic DNA, which can affect the quality of RNA.

2. Add 0.5 ml of isopropanol per ml of TRIzoI.

Note: If total RNA is expected to be less than 50ng, add in 0.5ul of glycogen as a carrier.

3. Shake and incubate for lOmin at room temperature.

4.4℃, centrifuge at 12OOOg for lOmin.

5. Carefully remove the supernatant c

6. Wash the RNA precipitate with 75% ethanol (Iml per ml of TRIzol).

7. Shake.

8. Centrifuge at 7500 g for 5 min at 4°C.

9. Carefully remove the supernatant.

10. Dry the precipitate in air for 15 min.

Note: The precipitate must not be too dry. Do not use a centrifugal vacuum precipitation device or the precipitate will be difficult to dissolve.

11. Dissolve the precipitate in DEPC-treated redistilled (distilled) water.

Note: If all RNA is used directly for cDNA synthesis, dissolve it with 11 ul ddH20.

12. Measure the OD260/280 values and calculate the RNA yield.

13. Preserve RNA by adding 0.1x volume of 3 mol/L NaAc and 2x volume of anhydrous ethanol. Dispense RNA into 2.5ug min per portion at -70°C.

5. RNA quantification

1. Remove a portion of RNA (e.g., one-tenth of the total RNA yield) and add DEPC-treated redistilled water to a final volume of 0.5 ml (if using a 0.5 ml quartz cuvette) or Iml (Iml quartz cuvette).

Note: At least 2ug of RNA is required.

2. Measure the OD of 260/280.

3. Determine RNA yield and quality.

A 260 OD of 1 is equivalent to 40 yg/ml of RNA

A 260/280 OD ratio of 2 indicates that the RNA is pure.

Note: An OD ratio of 260/280 less than 2 indicates the presence of protein and/or genomic DNA in the product, especially the presence of genomic DNA will cause false positive results.

6. DNA enzyme treatment

To prevent possible contamination of genomic DNA, the samples were treated with RNAase-free DNA enzymes.

1. Solubilize the RNA precipitate with DNAzyme buffer for IX.

2. Add 5U of DNAzyme.

3. Incubate at 37℃ for 15 min.

4. Increase the volume with DEPC-treated redistilled (distilled) water, e.g. up to 300 ul.

5. Add equal volumes of phenol/chloroform/isoamyl alcohol.

6. Shake vigorously.

7. Centrifuge at 13OOOg for 4 min at 4°C.

8. Transfer the upper aqueous phase into a clean Eppendorf tube and measure the quality and quantity of the RNA sample using a spectrophotometer.

9. The precipitated RNA is stored at -70°C.

7. RNA quality control

The quality of RNA was detected by 1% agarose gel electrophoresis with EB staining. Use sterilized buffer, glassware and 0.5 mol/L NaOH to clean the electrophoresis tank before electrophoresis.

The 18S and 28SrRNA bands should be clearly seen.

If there are no bands or the bands are very faintly stained and the majority of the EB staining is at the bottom of the gel, the RNA is degraded and cannot be used for differential display

cDNA synthesis 1. General principles

Four different primers [i.e., (E)T12MA, (E)T12 MG, (E)T12MT, (E)T12MC] were used for each RNA to perform four different cDNA synthesis reactions. Moreover, a negative control was set for each primer.

Therefore, 8 cDNA synthesis reactions are required for each RNA sample.

2.RNA denaturation

1. Add 2.5 fxg of total RNA and DEPC-treated redistilled (distilled) water to a total volume of 10 ul.

2. add 2ul (25umol/L) of primer according to DD type.

3. mix well.

4. Incubate at 70℃ for lOmin.

5. Place directly on ice.

Note: Incubation at 70°C will destroy the tertiary structure of the mRNA molecule. If the tube is moved from 70X: to room temperature, the RNA will anneal again, reducing the synthesis of full-length cDNA molecules.

6. add on ice:

-4 ul 5X first strand buffer

-2 ul 0.1 mol/L DTT

I ul dNTPs (10 mmol/L)

-Total 19 ul

7. Mix well and place the tubes in a 25°C water bath.

8. Incubate for lmin.

9. Add 1 ul reverse transcriptase (200 units) or 1 ul redistilled water as control.

10. Mix and incubate for lOmin.

11. Place the test tube in a 42°C water bath.

12. Incubate for another 50 min.

13. Incubate at 70°C for IOmin to heat inactivate the reverse transcriptase.

14. Centrifuge for 10s to remove the agglutinated water attached to the tubes.

15. Store the cDNA samples at 4℃.

IIIA.DD-PCR 1. General Principles

Use a tip with a filter membrane for all PCR-related pipetting steps.

2. Programs

Dilute the 20 fxl cDNA sample to 100 Ml with redistilled water. take 4 dilutions in the PCR reaction.

Note: If this is the first time the DI>PCR reaction has been operated, it will be necessary to optimize the reaction conditions for PCR. For example, j using a series of different dilutions of cDNA can determine the optimal concentration of template in the DD-PCR reaction.

3.PCR reaction

Note: A pipetting scheme should be designed before proceeding.

Add the following ingredients:

-4 ul cDNA

4 ul 10X buffer solution (including: Taq enzyme)

2 ul Mgcl2

-2 ul dNTPs (1 mmol/L)

-4 ul DIG-labeled T12MN (2.5/umol/L)

-4DD-digo (5/umol/L)

-18 ul ddH2O

-2 ul Taq enzyme (0.2 U/ul)

Total 40 ul

Caution:

1. In some cases the 10X buffer already contains MgCl2, in which case no MgCl2 is added and 20 ul ddH2 〇 should be added instead of 18 ul.

2. It is recommended that a mixture of 10x buffer, MgC12, ddH2, and specific primers be added together beforehand.

3. If the thermocycler does not have a heated lid, a drop of mineral oil can be added to prevent volatilization.

4. Preheat the PCR sample to 60°C before adding Taq enzyme. Annealing conditions for primers at room temperature are not stringent. Since the enzyme is mildly active at room temperature, too many cDNA fragments are produced and results are not reproducible.

4. PCR Process

A 94 ℃, 3 min; 37 ℃, 5 min; 72 ℃, 1 min

Then: 95℃,30s;38℃,2.5 min;72℃,45s; cycle 39 times

-72°C,5 min

Note: Reaction conditions are PCR instrument dependent. Our experimental conditions were optimized by Biometra and it is recommended to try to change these conditions for DD reactions.

5. Gel electrophoresis

A direct blotting device (GATC1500) is used to separate cDNA fragments produced by DD-PCR by size. This device is specially designed for the separation and detection of high-tech labeled DNA molecules directly imprinted on a nylon membrane during PAA gel electrophoresis. The DNA fragments on the membrane can be visualized by staining with anti-DIG antibody. The advantage of this system is that it is non-radioactive. Furthermore, it has a wide separation range of 10-800 bp, compared to the classical PAA gel electrophoresis of 10-300 bp or 150-500 bp, which reduces the amount of PAA gel needed. Since this procedure is designed specifically for this device and is explained in detail, we will only describe some general notes here.

1. For accurate comparison of cDNA fragments, PCR products amplified with the same primers are spotted next to each other (Figure 2-2).

2. The generation of false positives is a major problem with DD. To avoid picking up false positives for further analysis, increase the N-number, i.e., for each treatment group isolate multiple RNA samples for cDNA synthesis as well as PCR, and electrophoresis the samples from the same group next to each other. On the gel, only identified up- or down-regulated cDNA fragments appear for each sample in the treatment group for further analysis. By following this principle, there will be no false positive results.

III B. EDD-PCR

20cDNA samples were diluted to 25 ml,14 using ddH20 for PCR amplification. It is recommended that a series of different dilutions of cDNA be used to determine the optimal concentration of template when first operating EDI>PCR.

Note: PCR primers should be the same as those used for cDNA synthesis. For example, when 5'-[FAM]E1T12 MG is used for cDNA synthesis, 5'_[FAM]E1T12 MG should also be used for PCR.

1. PCR reaction

Add in the test tube:

-I ul cDNA

-2 ul 10XPCR Buffer Solution II (purchased with enzyme)

-1.6 ul Mgcl2 (25 umol/L)

-3.5 ul dNTPs (0.5 umol/L)

2 ul fluorescent EDD primers (2umol/L)

-2 ul BlDD primer (2umol/L)

-0.4 ul BSA (10 mg/ml)

-7.1 ul ddH2O

-0.4 ul Amplitaq Gold (5U/ul)

Total 20ul.

Caution:

1. Prepare a mixture of the components used in each group of reactions, including buffer, MgCl2, dNTPs, BSA, H2O, and Amplitaq Gold. Amplitaq Gold is only activated by incubation for IOmin at 95 "C, so a hot start is not necessary.

2. For each primer pair a negative control (plus H20) was set up. As mentioned earlier, EDD-PCR is very sensitive and can easily cause false positives.

2. PCR process

95°C, 1Omin.

-95°C,30s;38°C,2 min;72°C,2 min; cycle 4 times

-95°C,30s;60t:lmin;72°C,1.3 min; cycle 30 times

Note: This "complex" PCR process can be performed at low annealing temperatures (38°C) during the first 5 cycles, allowing the BlDD primer to anneal with many different cDNA molecules. cDNA molecules that are moderately homologous to the BlDD primer will anneal under these conditions. Then, by increasing the annealing temperature to 60t: it is possible to amplify only the cDNA molecules that started the reaction in the first 5 cycles, thus avoiding false positives caused by subsequent random start reactions.

3. Gel electrophoresis of incandescent-labeled EDD-cDNA fragments

We analyzed cDNA fragments generated by fluorescent EDD-PCR using an automated DNA sequencer (Figs. 2-3). Using the GATC device, the advantage of this reaction system is that it does not require the detection of DNA molecules by radioactivity, and fragments with a wide range of lengths (10-1200 bp) can be separated, thus requiring fewer PAA-gels. Another outstanding advantage of using an automated DNA sequencer is the possibility of automated analysis of DNA sequences with specialized software and digital storage of EDD-data. Since automated DNA sequencers require detailed operating instructions, these are beyond the scope of this book and will not be described further here.

4. General Principles

1. Non-radiolabeling is used in this protocol. As labeling methods do not normally affect the activity of enzymes (e.g., reverse transcriptase or Tag polymerase), this scheme applies equally to other markers, including radiolabeling.

2. Isolation of the target EDD-PCR fragments:

(1) Add (32P-a)dATP, and do 4~6 cycles. Then the fragments can be separated by conventional PAA gel electrophoresis.

(2) Add DIG-labeled primer and do another 4-6 cycles. The resulting fragments are then blotted onto a reaction membrane. Half of the membrane can be stained with anti-DIG antibody to localize the fragments, and the other half will be cut off with the corresponding target fragments.'' Boil the membrane in boiling water for lOmin, elute the DNA, and perform 20~25 cycles of PCR reaction. Identify and analyze the resulting fragments by agarose gel electrophoresis and clone them, which should be avoided in this process:

Use HykmdN+, because this nylon membrane has a very strong ability to bind DNA and the DNA will not be released after boiling.

Cross-link DNA after membrane blotting, which also prevents the elution of DNA after boiling.

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